N-acyldipeptide Derivatives and Their Uses

ABSTRACT

Methods of treating aging related skin changes and of increasing skin thickness with topical administration of N-acyldipeptide derivatives are described. Compositions comprising N-acyldipeptide derivatives, are therapeutically effective for increasing skin thickness, and for treating extrinsic and intrinsic aging and aging related skin changes, such as fine lines, wrinkles, photoaging, hyperpigmentation, laxity, age spots, lentigines, mottled skin, and cellulite.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/407,474, filed Jan. 17, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/171,601, filed Jun. 2, 2016, which is acontinuation of U.S. patent application Ser. No. 14/718,317, filed May21, 2015, now U.S. Pat. No. 9,370,546, issued Jun. 21, 2016, which is adivisional of U.S. patent application Ser. No. 14/354,711 filed Apr. 28,2014, now U.S. Pat. No. 9,067,969, issued Jun. 30, 2015, which is aSection 371 of International Application No. PCT/US2012/062715, filedOct. 31, 2012, which was published on May 2, 2013, under InternationalPublication No. WO 2013/063615 A2, which claims priority under 35 U.S.C.§ 119(e) to Provisional Patent Application No. 61/552,751, filed on Oct.28, 2011, Provisional Patent Application No. 61/554,724, filed on Nov.2, 2011, and Provisional Patent Application No. 61/582,675, filed onJan. 3, 2012, and the disclosures of which are incorporated by referenceherein in their entireties.

FIELD OF THE INVENTION

The embodiments described herein relate to compositions and uses of thecompositions comprising N-acyldipeptide derivatives for systemic ortopical administration to a mammal to alleviate or improve diseases,symptoms or syndromes associated with tumors, cancers, immune, nervous,vascular, musculoskeletal or cutaneous system, or other tissues andsystems.

BACKGROUND OF THE INVENTION

It was reported that a peptide ester, Asp-Phe-OMe (Aspartame), hadsucrose-like sweet taste, but all other related peptides such asAla-Phe-OMe, His-Phe-OMe, etc. had bitter taste. See Mazur et al.“Structure-Taste Relationships of Some Small Peptides” in PEPTIDES:Chemistry and Biochemistry by Boris Weinstein and Saul Lande (MarcelDekker, Inc. New York), 1970, 175-180. In Handbook of Neurochemistry andMolecular Neurobiology 3^(rd) Ed. “Amino Acids and Peptides in theNervous System” by Oja et al. Springer Science 2007, page 401-411,Reichelt describes in “Low Molecular Weight Peptides” endogenouspeptides. The endogenous dipeptide described is N-pyroglutamyl (N-PyroE)peptide, N-PyroE-His-Pro-NH₂. In Enzyme and Microbial Technology 45(2009) 457-462, entitled “Availability of tyrosine amide forα-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides”Narai-Kanayama et al. describe that oligo-tyrosine peptides such asTyr-Tyr having angiotensin I-converting enzyme inhibitory activity canbe synthesized by an α-chymotrypsin-catalyzed reaction with N—Ac-Tyr-OEtand Tyr-OEt or Tyr-NH₂ as starting substances. Kinetic analysis showedthat assumed products, N—Ac-Tyr-Tyr-OEt or N—Ac-Tyr-Tyr-NH₂ did nothydrolyze to yield the desired Tyr-Tyr. There is no description orreport about the composition or use of the composition comprisingN-acyldipeptide derivatives of the present invention.

BRIEF SUMMARY OF THE INVENTION

It has been discovered in the present invention that compositionscomprising the N-acyldipeptide derivatives are therapeutically effectivefor topical or systemic administration to alleviate or improveconditions, disorders, diseases, symptoms or syndromes associated withtumors, cancers, immune, nervous, vascular, musculoskeletal or cutaneoussystem, or other tissues or systems in a subject.

According to one embodiment of the present invention, theN-acyldipeptide derivative is represented by the following genericFormula (I):

R₁-AAB-AAC-R₂  Formula (I)

or an isomer, free acid, base, salt, lactone, amide, hydrazide, or esterthereof, wherein R₁ is an acyl radical having up to 19 carbon atoms; AABis an amino terminal amino acid residue selected from any amino acid;AAC is a carboxyl terminal amino acid residue selected from any aminoacid; R₂ is OR₃, NHR₄ or NHNHR₅; R₃ is H, an alkyl, aralkyl or arylradical having up to 19 carbon atoms; R₄ or R₅ is independently H, OH,an alkyl, aralkyl, aryl or acyl radical having up to 19 carbon atoms; aside chain of each of AAB and AAC optionally and independently has anextra functional radical selected from the group consisting of OH, SH,NHCONH₂, NHC(═NH)NH₂, NH₂, COOH, CONH₂, imidazolyl, pyrrolidinyl andindolyl; and the H or OH of the extra functional radical is optionallysubstituted by NH₂, an acyl, alkyl, aralkyl, or aryl radical having upto 19 carbon atoms.

In a preferred embodiment, AAB is an amino terminal amino acid residueselected from the group consisting of Ala, βAla, Abz, Asn, Cre, Cys,Dopa, Gly, Gln, Glu Gaba, His, Hpg, Ile, Leu, Pgly, Phe, Pro, Ser, Trp,Tyr, and Val; and AAC is a carboxyl terminal amino acid residue selectedfrom the group consisting of Ala, Cre, Cys, Dopa, Gly, His, Hpg, Ile,Leu, Lys, Pgly, Phe, Pro, Trp and Tyr.

In another aspect, an embodiment of the present invention relates to acomposition comprising a therapeutically effective amount of anN-acyldipeptide derivative of the invention and a pharmaceutically orcosmetically acceptable carrier for topical or systemic administrationto a subject.

In yet another aspect, an embodiment of the present invention relates toa method of alleviating or improving conditions, disorders, diseases,symptoms or syndromes associated with tumors, cancers, immune, nervous,vascular, musculoskeletal or cutaneous system, or other tissues orsystems in a subject. The method comprises topically or systemicallyadministering to the subject a composition of the present invention.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

DESCRIPTION OF THE INVENTION

Various publications are cited or described in the background andthroughout the specification; each of these references is hereinincorporated by reference in its entirety. Discussion of documents,acts, materials, devices, articles or the like which has been includedin the present specification is for the purpose of providing context forthe present invention. Such discussion is not an admission that any orall of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. Allpublications cited herein are incorporated by reference as if set forthfully herein. It must be noted that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

Common or certain knowledge, scientific and medical terminologies can bereadily found via internet, textbooks of chemistry, biochemistry,medicinal chemistry, pharmacology, dermatology and general medicine. Thefollowing are some examples. Robert K. Murray et al. eds. “Harper'sIllustrated Biochemistry” 26^(th) edn. Vol. I-II, McGraw Hill, 2003.Laurence L. Brunton et al. eds. “Goodman & Gilman's The PharmacologicalBasis of Therapeutics” 12^(th) edn. McGraw Hill Medical, 2011. KlausWolff et al. eds. “Fitzpatrick's Dermatology in General Medicine” 7^(th)edn. Vol. I-II, McGraw Hill Medical, New York, 2008. Tony Burns et al.eds. “Rook's Textbook of Dermatology” 8^(th) edn. Vol. I-IV,Wiley-Blackwell, 2010. Anthony S. Fauci et al. eds. “Harrison'sPrinciples of Internal Medicine” 17^(th) edn, McGraw Hill Medical, NewYork, 2008.

An amino acid is an organic acid having one or more than one alkalineradical such as amino, guanidino, imino, or hydrazine radical attachedat any carbon atom other than carbon one. There are 20 common aminoacids which are represented by chemical names, such as “glycine”, orabbreviated symbols such as three letters, “Gly” or one letter “G”. Inthis disclosure, three letters will be used. Except glycine, all othercommon amino acids have stereoisomers, i.e., enantiomer, D or L form.The amino acids in most natural peptides and proteins are all in L-form.Some D-form amino acids are produced by microorganisms or present inantibiotics, and have inhibitory or antagonistic actions. For example,D-alanine, D-aspartic acid, and D-glutamic acid are present in bacterialcell walls, and D-glutamic acid, D-aspartic acid and D-phenylalanine arepresent in the antibiotic bacitracin. An uncommon amino acid is an aminoacid that is not a common amino acid. Examples of uncommon amino acidsinclude, but are not limited to, β-alanine and taurine. The uncommonamino acids can exist in D or L form.

The three letter symbols used for the 20 common amino acids are asfollows:

alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn),cysteine (Cys), glycine (Gly), glutamic acid (Glu), glutamine (Gln),histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Inthis disclosure, not all the common amino acids may be included in theN-acyldipeptide derivatives of the present invention.

The letter symbols used for uncommon amino acids are as follows:β-alanine (βAla), 4-aminobenzoic acid (Abz), aminobutanoic acid (Aba),γ-aminobutanoic acid (Gaba), β-aminoisobutanoic acid (Baia),5-aminolevulinic acid (All), alliin (Ali), aminoadipic acid (Aaa),aminopimelic acid (Apa), 3-aminotyrosine (Atyr), canavanine (Cav),canaline (Can), ciliatine (Cil), cysteic acid (Cya), cysteine sulfinicacid (Csa), citruline (Cit), creatine (Cre), creatinine (Cm),2,3-diaminosuccinic acid (Dsa), 2,4-diaminobutanoic acid (Dba),2,3-diaminopropanoic acid (Dpa), 3,4-dihydroxyphenyl-alanine (Dopa),3,5-diiodotyrosine (Dtyr), homoarginine (Har), homoserine (Hser),homocysteine (Hcys), homocitrulline (Hcit), 5-hydroxylysine (Hlys),4-hydroxyproline (Hpro), 2-hydroxy-4-aminobutanoic acid (Haba),3-hydroxy-4-aminobutanoic acid (Hyba), 4-hydroxyornithine (Horn),4-hydroxyaspartic acid (Hasp), 4-hydroxyphenyl-glycine (Hpg),3-iodotyrosine (Ityr), lanthionine (Lan), β-lysine (βLys),α-methylalanine (Mala), β-methylaspartic acid (Mas), 4-methylproline(Mpro), 2-methylserine (Mser), N-methylhistidine (Mhis), ornithine(Orn), phenylglycine (Pgly), 3-phenylserine (Pser), sarcosine (Sar),S-allyl-cysteine (Sac), theanine (The), thyroxine (Thy),3,5,3′-triiodothyronine (Tth), and taurine (Tau). In this disclosure,not all the uncommon amino acids may be included in the N-acyldipeptidederivatives of the present invention.

The terms and abbreviations that can be used are as follows: acetyl, Ac;benzoyl, Bz; benzyl, Bzl; diphenylmethyl, Dpm; benzyl ester, OBzl;benzyloxycarbonyl, Z; t-butyl ester, OtBu; t-butyl, tBu; ethyl ester,OEt; formyl, For; hexyl ester, OHex; methyl ester, OMe; propanoyl, Pr;pyroglutamyl, Pyro; phenylacetyl, PhAc; and trityl, Trt.

A peptide bond, C(═O)NH, is a covalent bond formed between two aminoacid molecules when the carboxyl group on one amino acid reacts with theamino group of the other amino acid in a dehydration synthesis reaction.A dipeptide is a peptide that contains two amino acid residues.Theoretically, 400 different dipeptides can be formed from 20 commonamino acids,

and more than 5,000 different dipeptides can be formed from both thecommon and uncommon amino acids. The dipeptides can be further modifiedby substitutions, etc. Each dipeptide can have different chemical andphysical properties, and can have different biological andpharmacological actions.

When a particular group is “substituted”, that group can have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.

With reference to substituents or amino acid residues in a peptide, theterm “independently” means that when more than one of such substituentsor amino acid residues are possible, such substituents or amino acidresidues may be the same or different from each other.

As used herein, the term “subject” means any animal, preferably amammal, most preferably a human, to whom will be or has beenadministered compounds or topical formulations according to embodimentsof the invention. The term “mammal” as used herein, encompasses anymammal. Examples of mammals include, but are not limited to, cows,horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs,monkeys, humans etc., more preferably, a human.

In one embodiment, “treatment” or “treating” refers to an amelioration,prophylaxis or reversal of a disease or disorder, or at least onediscernible symptom thereof. In another embodiment, “treatment” or“treating” refers to an amelioration, prophylaxis, or reversal of atleast one measurable physical parameter related to the disease ordisorder being treated, not necessarily discernible in or by the mammal.In yet another embodiment, “treatment” or “treating” refers toinhibiting or slowing the progression of a disease or disorder, eitherphysically, stabilization of a discernible symptom, physiologically,e.g., stabilization of a physical parameter, or both. In yet anotherembodiment, “treatment” or “treating,” refers to delaying the onset of adisease or disorder.

In certain embodiments, compounds of interest are administered as apreventative measure. As used herein, “prevention” “preventing” refersto a reduction of the risk of acquiring a given disease or disorder.

As used herein, a “therapeutically effective amount” of a compound of anembodiment of the present invention means the amount of the compoundthat elicits the biological or medicinal response in a tissue system,animal or human that is being sought by a researcher, veterinarian,medical doctor or other clinician, which includes alleviation of thesymptoms of the disease or disorder being treated.

One skilled in the art will recognize that the therapeutically effectiveamount of a compound to be used in the instant invention can vary withfactors, such as the particular subject, e.g., age, diet, health, etc.,severity and complications and types of the symptom or disorder soughtto be treated or prevented, the formulation used, etc.

According to one embodiment of the present invention, theN-acyldipeptide derivative of the present invention is represented bythe following generic Formula (I):

R₁-AAB-AAC-R₂  Formula (I)

or an isomer, free acid, base, salt, lactone, amide, hydrazide, or esterthereof, wherein R₁ is an acyl radical having up to 19 carbon atoms; AABis an amino terminal amino acid residue selected from any amino acid;AAC is a carboxyl terminal amino acid residue selected from any aminoacid; R₂ is OR₃, NHR₄ or NHNHR₅; R₃ is H, an alkyl, aralkyl or arylradical having up to 19 carbon atoms; R₄ or R₅ is independently H, OH,an alkyl, aralkyl, aryl or acyl radical having up to 19 carbon atoms; aside chain of each of AAB and AAC optionally and independently has anextra functional radical selected from the group consisting of OH, SH,NHCONH₂, NHC(═NH)NH₂, NH₂, COOH, CONH₂, imidazolyl, pyrrolidinyl andindolyl; and the H or OH of the extra functional radical is optionallysubstituted by NH₂, an acyl, alkyl, aralkyl, or aryl radical having upto 19 carbon atoms. A typical acyl radical includes, but not limited to,acetyl (Ac), propanoyl (Pr), or benzoyl (Bz). A typical group attachedto the carboxyl terminal amino acid residue includes, but not limitedto, OH, OEt, NH₂, NHOH, or NHNH₂.

Preferably, AAB is an amino terminal amino acid residue selected fromthe group consisting of Ala, βAla, Abz, Asn, Cre, Cys, Dopa, Gly, Gln,Glu, Gaba, His, Hpg, Ile, Leu, Pgly, Phe, Pro, Ser, Trp, Tyr, and Val;and AAC is a carboxyl terminal amino acid residue selected from thegroup consisting of Ala, Cre, Cys, Dopa, Gly, His, Hpg, Ile, Leu, Lys,Pgly, Phe, Pro, Trp and Tyr.

Based on Formula (I), the N-acyldipeptide derivatives of the presentinvention can be divided according to the carboxyl terminal amino acid,which include, but are not limited to the following N-acyldipeptidederivatives:

1. N-Acetyldipeptide Derivatives

(a) N-Acetyl alanine dipeptide derivatives, including, but not limitedto: N—Ac-Ala-Ala-OH, N—Ac-Ala-Ala-OEt, N—Ac-Ala-Ala-NH₂,N—Ac-Glu-Ala-OH, N—Ac-Glu-Ala-NH₂, N—Ac—Ile-Ala-OH, N—Ac—Ile-Ala-NH₂,N—Ac-Leu-Ala-OH, N—Ac-Leu-Ala-NH₂, N—Ac—Val-Ala-OH, N—Ac—Val-Ala-NH₂,N—Ac-Glu-Ala-NH₂, N—Ac—Cys-Ala-NH₂, N—Ac-Dopa-Ala-NH₂.

(b) N-Acetyl glycine dipeptide derivatives, including, but not limitedto: N—Ac-β-Ala-Gly-NH₂, N—Ac-Asn-Gly-NH₂, N—Ac-Abz-Gly-NH₂,N—Ac—Cys-Gly-NH₂, N—Ac-Gaba-Gly-NH₂, N—Ac-Gly-Gly-NH₂, N—Ac-Gln-Gly-NH₂,N—Ac—His-Gly-NH₂, N—Ac—Ile-Gly-NH₂, N—Ac—Ile-Gly-OH, N—Ac-Leu-Gly-NH₂,N—Ac-Leu-Gly-OH, N—Ac—Pro-Gly-NH₂, N—Ac—Ser-Gly-NH₂, N—Ac-Tyr-Gly-NH₂,N—Ac—Val-Gly-NH₂, N—Ac—Val-Gly-OH.

(c) N-Acetyl tyrosine dipeptide derivatives, including, but not limitedto: N—Ac-Cys-Tyr-NH₂, N—Ac-Dopa-Tyr-NH₂, N,O,O′-Tri-Ac-Dopa-Tyr-NH₂,N,O,O′-Tri-Ac-Dopa-Tyr-OEt, N—Ac—Hpg-Tyr-NH₂, N—Ac-Tyr-Tyr-OH,N—Ac-Tyr-Tyr-OEt, N—Ac-Tyr-Tyr-NH₂, N—Ac—Ile-Tyr-OH, N—Ac—Ile-Tyr-NH₂,N—Ac-Leu-Tyr-OH, N—Ac-Leu-Tyr-NH₂, N—Ac—Val-Tyr-OH, N—Ac—Val-Tyr-NH₂,N—Ac-Tyr-Tyr-NHNH₂, N—Ac-Tyr-Tyr-NHNHAc.

(d) Other N-acyldipeptide derivatives, including, but not limited to:N—Ac-Arg-Cre-NH₂, N—Ac—Cre-Cre-NH₂, N—Ac—Cre-Cre-OH, N—Ac—Ser-Cre-NH₂,N—Ac-Tyr-Cre-NH₂, N—Ac—Cre-Tyr-NH₂,N—Ac-Cre-Ala-NH₂, N—Ac—Cys-Cys-NH₂,N—Ac—Cys-Cys-OH, N,O,O′-Tri-Ac-Dopa-Cys-OH, N,O,O′-Tri-Ac-Dopa-Cys-NH₂,N—Ac—Cys-O,O′-Di-Ac-Dopa-OH, N—Ac—Cys-O,O′-Di-Ac-Dopa-OEt,N—Ac-Dopa-Dopa-NH₂, N—Ac-Tyr-Dopa-NH₂, N—Ac-Glu-Glu-NH₂,N—Ac-βAla-His-NH₂, N,N′-Di-Ac-βAla-His-NH₂, N—Ac-βAla-1-MHis-NH₂,N—Ac-Ala-Ile-NH₂, N—Ac—Ile-Ile-NH₂, N—Ac-Asp-Glu-NH₂, N—Ac-Gly-Gln-NH₂,N—Z-Tyr-Gly-NH₂, N—Ac-Gaba-His-NH₂, N—Ac-Leu-Leu-NH₂, N—Ac-Gly-Ile-NH₂,N—Ac-Tyr-Hpg-NH₂, N—Ac-βAla-His-OEt, N—Ac-Phe-Phe-NH₂, N—Ac-Trp-Trp-NH₂,N—Ac—Pgly-Pgly-NH₂, N—Ac—Hpg-Hpg-NH₂, N—Ac-Tyr-Tyr-NHOH,N—Ac—Val-Ala-NHOH, N—Ac-Glu-Lys-NH₂, and N—Ac-Gln-Lys-NH₂.

2. N-Propanoyldipeptide Derivatives.

(a) N-Propanoyl alanine dipeptide derivatives, including, but notlimited to: N—Pr-Ala-Ala-OH, N—Pr-Ala-Ala-OEt, N—Pr-Ala-Ala-NH₂,N—Pr—Ile-Ala-OH, N—Pr—Ile-Ala-NH₂, N—Pr-Leu-Ala-OH, N—Pr-Leu-Ala-NH₂,N—Pr—Val-Ala-OH, N—Pr—Val-Ala-NH₂, N—Pr-Glu-Ala-NH₂.

(b) N-Propanoyl glycine dipeptide derivatives, including, but notlimited to: N—Pr-Gly-Gly-OH, N—Pr-Gly-Gly-OEt, N—Pr-Gly-Gly-NH₂,N—Pr-Ala-Gly-NH₂, N—Pr-βAla-Gly-NH₂, N—Pr-Asn-Gly-NH₂, N—Pr—Cys-Gly-NH₂,N—Pr-Gln-Gly-NH₂, N—Pr-Gaba-Gly-NH₂, N—Pr—His-Gly-NH₂, N—Pr—Ile-Gly-OH,N—Pr—Ile-Gly-NH₂, N—Pr-Leu-Gly-OH, N—Pr-Leu-Gly-NH₂, N—Pr—Pro-Gly-NH₂,N—Pr—Ser-Gly-NH₂, N—Pr-Tyr-Gly-NH₂, N—Pr—Val-Gly-OH, N—Pr—V al-Gly-NH₂.

(c) N-Propanoyl tyrosine dipeptide derivatives, including, but notlimited to: N—Pr-Tyr-Tyr-OH, N—Pr-Tyr-Tyr-OEt, N—Pr-Tyr-Tyr-NH₂,N—Pr—Ile-Tyr-OH, N—Pr—Ile-Tyr-NH₂, N—Pr-Leu-Tyr-OH, N—Pr-Leu-Tyr-NH₂,N—Pr—Val-Tyr-OH, N—Pr—Val-Tyr-NH₂, N—Pr-Tyr-Tyr-NHNH₂,N—Pr-Tyr-Tyr-NHNHPr, N—Pr-Dopa-Tyr-NH₂, N—Pr—Cys-Tyr-NH₂.

(d) Other N-propanoyldipeptide derivatives, including, but not limitedto: N—Pr-Glu-Glu-OH, N—Pr-Glu-Glu-OEt, N—Pr-Glu-Glu-NH₂,N—Pr-Glu-Ala-OH, N—Pr-Glu-Ala-NH₂, N—Pr-βAla-His-NH₂,N—Pr-βAla-1-MHis-NH₂,N—Pr-Ala-Ile-NH₂, N—Pr-Asp-Glu-NH₂,N—Pr-Gly-Gln-NH₂, N—Pr-Gaba-His-NH₂, N—Pr-Leu-Leu-OH, N—Pr-Leu-Leu-OEt,N—Pr-Leu-Leu-NH₂, N—Pr-Gly-Ile-NH₂, N—Pr—Ile-Ile-OH, N—Pr—Ile-Ile-NH₂,N—Pr-βAla-His-OEt, N—Pr—Cys-Cys-NH₂, N—Pr-Dopa-Dopa-NH₂,N—Pr-Dopa-Cys-NH₂, N—Pr—Cys-Dopa-NH₂, N—Pr-Phe-Phe-NH₂,N—Pr-Trp-Trp-NH₂, N—Pr-Glu-Lys-NH₂, and N—Pr-Gln-Lys-NH₂.

The preferred bioactive dipeptide derivative of the present invention isselected from the group consisting of N—Ac-Ile-Ala-NH₂,N—Ac-Leu-Ala-NH₂, N—Ac—Val-Ala-NH₂, N—Ac—Cys-Cys-NH₂, N—Ac-Tyr-Cys-NH₂,N—Ac-Dopa-Dopa-NH₂, N—Ac—Ile-Gly-NH₂, N—Ac-Leu-Gly-NH₂,N—Ac—Val-Gly-NH₂, N—Ac-Tyr-Tyr-NH₂, N—Ac—Cys-Tyr-NH₂, N—Ac—Ile-Ala-OH,N—Ac-Leu-Ala-OH, N—Ac—Val-Ala-OH, N—Ac—Cys-Cys-OH, N—Ac-Tyr-Cys-OH,N—Ac-Dopa-Dopa-OH, N—Ac—Ile-Gly-OH, N—Ac-Leu-Gly-OH, N—Ac—Val-Gly-OH,N—Ac-Tyr-Tyr-OH, N—Ac—Cys-Tyr-OH, N—Pr-Ile-Ala-NH₂, N—Pr-Leu-Ala-NH₂,N—Pr—Val-Ala-NH₂, N—Pr—Cys-Cys-NH₂, N—Pr-Tyr-Cys-NH₂,N—Pr-Dopa-Dopa-NH₂, N—Pr-Ile-Gly-NH₂, N—Pr-Leu-Gly-NH₂,N—Pr—Val-Gly-NH₂, N—Pr-Tyr-Tyr-NH₂, N—Pr—Cys-Tyr-NH₂, N—Pr—Ile-Ala-OH,N—Pr-Leu-Ala-OH, N—Pr—Val-Ala-OH, N—Pr-Cys-Cys-OH, N—Pr-Tyr-Cys-OH,N—Pr-Dopa-Dopa-OH, N—Pr—Ile-Gly-OH, N—Pr-Leu-Gly-OH, N—Pr—Val-Gly-OH,N—Pr-Tyr-Tyr-OH, and N—Pr-Cys-Tyr-OH.

The more preferred bioactive dipeptide derivative of the presentinvention is selected from the group consisting ofN—Ac-Ile-Ala-NH₂,N—Ac-Leu-Ala-NH₂,N—Ac-Val-Ala-NH₂, N—Ac—Cys-Cys-NH₂,N—Ac-Tyr-Cys-NH₂, N—Ac-Dopa-Dopa-NH₂, N—Ac-Leu-Gly-NH₂,N—Ac-Tyr-Tyr-NH₂, N—Ac—Cys-Tyr-NH₂, N—Ac—Ile-Ala-OH, N—Ac-Leu-Ala-OH,N—Ac—Val-Ala-OH, N—Ac—Cys-Cys-OH, N—Ac-Tyr-Cys-OH, N—Ac-Dopa-Dopa-OH,N—Ac—Ile-Gly-OH, N—Ac-Leu-Gly-OH, N—Ac—Val-Gly-OH, N—Ac-Tyr-Tyr-OH, andN—Ac—Cys-Tyr-OH.

The most preferred bioactive dipeptide derivative of the presentinvention is selected from the group consisting of N—Ac-Ile-Ala-NH₂,N—Ac-Leu-Ala-NH₂, N—Ac—Cys-Cys-NH₂, N—Ac-Tyr-Cys-NH₂,N—Ac-Dopa-Dopa-NH₂, N—Ac-Leu-Gly-NH₂, N—Ac-Tyr-Tyr-NH₂, andN—Ac-Cys-Tyr-NH₂.

N-acyldipeptide derivatives according to embodiments of the presentinvention can be made by any method known to those skilled in the art inview of the present disclosure.

Chemical and physical properties, biological functions and therapeuticeffects of a peptide depend exclusively on the nature and sequence ofamino acid residues, and different amino acid residues or a differentamino acid sequence may result in a completely different peptide. Inaddition to chemical and physical properties, biological functions andtherapeutic effects of a peptide are also changed when the functionalgroups of such peptide are modified by substitution. In most cases, thebioactive dipeptide derivative of the present invention has differentand much improved chemical and physical properties, biological functionsand therapeutic effects as compared to an unmodified peptide.

A peptide is usually an amphoteric substance, having positive andnegative charges in the same molecule. A peptide normally cannotpenetrate the skin on topical application because of the tough stratumcorneum layer that acts as a permeation barrier. In general, an ionicsubstance or any substance with a molecular weight of more than 800daltons cannot readily penetrate the intact skin. The N-acyldipeptidederivatives of the present invention have the alkaline radical such asan amino group modified by acylation, so that they are no longeramphoteric in nature, and are readily bioavailable for penetrationand/or distribution to target tissues or sites for pharmacologicalactions by topical or systemic administration.

Another general aspect of the present invention relates to a method oftreating or preventing a disease, symptom or syndrome associated withimmune, tumors, cancers, nervous, vascular, musculoskeletal, cutaneoussystem, or other tissues and systems in a subject in need of thetreatment. The method comprises topically or systemically administeringto the subject a composition comprising a therapeutically effectiveamount of an N-acyldipeptide derivative according to an embodiment ofthe present invention and a pharmaceutically or cosmetically acceptablecarrier.

Conditions, disorders, symptoms and syndromes associated with the (A)tumors and cancers, (B) immune system, (C) nervous system, (D) vascularsystem, (E) musculoskeletal system, (F) cutaneous system, and (G) othertissues or systems that can be treated with a composition of the presentinvention are described as follows.

(A) Tumors and Cancers

Cancer is an unregulated proliferation of cells due to loss of normalcontrols, resulting in abnormal growth, lack of differentiation, localtissue invasion, and often, metastasis. Tumor is an abnormal growth ofcells or tissues which may be benign or malignant. Tumors or cancersthat can be treated with a composition of the present invention include,but are not limited to, actinic keratosis, adrenal cancer, basal cellcarcinoma, bladder cancer, brain tumor, breast cancer, cervical cancer,colon cancer, esophagus cancer, head and neck cancer, Hodgkin's disease,Kaposi's sarcoma, larynx cancer, leukemia, lung carcinoma, liver cancer,melanoma, multiple myeloma, mesothelioma, ovarian cancer, pancreaticcancer, prostate cancer, renal cancer, rectal cancer, stomach cancer,squamous cell carcinoma, thyroid cancer, testicular cancer, thyroidcancer, and uterine cancer. Breast cancer most often involves glandularbreast cells in the ducts or lobules, and can invade locally and spreadthrough lymph nodes and bloodstream, then to lungs, liver, bone, brainand skin. Lung carcinoma is a leading cause of lung cancer with symptomsof coughing, chest discomfort or pain, and weight loss. Liver cancer isusually hepatocellular carcinoma often resulting from liver cirrhosis.Pancreatic cancer, primarily ductal adenocarcinoma, has symptoms ofweight loss, abdominal pain, and jaundice. Brain tumors such as gliomas,medulloblastomas and ependymomas can have symptoms of headache, pain,edema, etc.

The development and growth of tumors and cancers can be due to derangedimmune system even though the tumors or cancers may be caused bymutations.

(B) Immune System

The immune system, very similar to organs such as liver, kidney andthyroid, is composed of specialized cells that play a vital role in hostdefense. These cells include leukocytes (white blood cells) anddendritic cells. The leukocytes are divided into granulocytes (65%),specific granules in the cytoplasm such as neutrophils, eosinophils, andbasophils; and agranulocytes, no specific granules in the cytoplasm suchas lymphocytes (25-35%) and monocytes (5-10%). The lymphocytes aresubdivided into B lymphocytes (antibody production) and T lymphocytes(foreign agent and tissue destruction). The monocyte can migrate fromblood to tissue, and become a macrophage. The dendric cell is derivedfrom bone marrow and is critical in activation and priming of thelymphocyte.

Deranged immune system can cause the following disorders:

(1) Rheumatic, connective tissue or collagen diseases. These diseasesinclude, but are not limited to, systemic lupus erythematosus,rheumatoid arthritis, seronegative spondylarthritides (ankylosingspondylitis), Sjogren's syndrome (keratoconjunctivitis sicca,xerostomia), systemic sclerosis, polymyositis and dermatomyositis.(2) Endocrine autoimmune diseases. These diseases include, but are notlimited to, Type 1 diabetes, autoimmune thyroid disease such as Graves'disease and Hashimoto's thyroiditis, and Addison's disease.(3) Liver diseases. These diseases include, but are not limited to,autoimmune hepatitis, sclerosing cholangitis, biliary cirrhosis, viralhepatitis including hepatitis A, hepatitis B, and hepatitis C.(4) Gastrointestinal diseases. These diseases include, but are notlimited to, mucosal disorder, atrophic gastritis, pernicious anemia,inflammatory bowel disease, and allergic food reactions.(5) Immune mediated nephritis and vasculitis. These diseases include,but are not limited to, glomerulonephritis, Wegener's granulomatosis,microscopic polyarteritis, and cryoglobulinanemia.(6) Immune mediated skin diseases. These diseases include, but are notlimited to, psoriasis, vitiligo, bullous pemphigoid, pemphigus vulgaris,and pemphigus foliaceus.(7) Immune mediated diseases of nervous system and eye. These diseasesinclude, but are not limited to, multiple sclerosis, Guillain-Barresyndrome, myasthenia gravis, Lambert-Eaton syndrome, stiff man syndrome,keratitis, keratoconjunctivitis sicca, scleritis, episcleritis, anduveitis.(8) Human immunodeficiency virus (HIV) and acquired immune deficiencysyndrome (AIDS). HIV is a member of retrovirus family, with asingle-stranded RNA genome. Such RNA genome can encode the enzymereverse transcriptase, capable of transcribing viral RNA into DNA, andallowing the virus to integrate into the host cell genome. During theinitial stage of infection, the virus targets memory CD4 T lymphocytesas a receptor, and depletes CD4 T cells from gut and peripheral lymphnodes. The immunity from B lymphocytes, dendritic cells and macrophagesis also weakened. The vaccine remains the best hope of controlling HIVinfection, however, there are numerous issues to be resolved for aneffective, inexpensive and safe immunization against HIV infection. Thechallenging issues are (a) the virus can survive and be transmittedwithin a host and between hosts in extracellular form, as blood bornevirus particles, and also in intracellular form hidden within infectedhost cells, (b) the virus copies its genome into host cells, and liveattenuated virus vaccine may pose a safety issue, (c) the virus hasmultiple strains and a very high mutability which is challenging for avaccine using fixed virus sequences, and may not be effective for otherstrains, (d) there is no small animal model existing for HIV infection,and the efficacy studies carried out for non-human primates are ratherexpensive.

Other deranged immune system may also involve the growth and spread(metastasis) of tumors and cancers.

(C) Nervous System

The conditions, disorders, symptoms and syndromes associated with thenervous system include, but are not limited to, the following conditionsor disorders, which may present as indicated, or otherwise: (1) dementiaand Alzheimer's disease: progressive loss of memory, shrinkage andatrophy of cerebral cortex, tangles of fibers in nerve cells, senileplaques of (3-amyloid, decreased choline acetyltransferase enzyme; (2)carpal tunnel syndrome: weakness, pain, tingling, numbness, burning inpalm and fingers; (3) encephalitis: inflammation of the brain; (4)headache: migraine, expansion of blood vessels pressing on nerves orconstriction blocking blood supply, inflammation, muscle contraction toface, neck or scalp; (5) meningitis: infection of spinal fluid andmeninges; (6) neuralgia: nerve pain, peripheral neuropathy, sciatica,shingles, trigeminal neuralgia; (7) Parkinson's disease: tremors inlimbs, muscular rigidity; (8) amnesia: loss of memory and inability toform new memory; and (9) others, such as ataxia, Bell's palsy, epilepsy,multiple sclerosis, myasthenia gravis, narcolepsy, paralysis and rabies.

Alzheimer's disease causes progressive cognitive deterioration and ischaracterized by senile plaques of β-amyloid deposits, neurofibrillarytangles in the cerebral cortex and subcortical gray matter, andcurrently there is no cure.

Parkinson's disease is an idiopathic, slowly progressive, degenerativecentral nervous system (CNS) disorder characterized by resting tremor,muscular rigidity, slow and decreased movement, and posturalinstability, and currently there is no cure.

(D) Vascular System

The vascular conditions, reactions and disorders that can be treatedwith a composition of the present invention include, but are not limitedto, acanthosis nigricans, acrocyanosis, actinic cheilitis, actinicprurigo, dermatitis, dermatosis, dermographism, dyshidrosis, drugeruptions, inflammation, or eczema, erythema, erythema migrans,erythrocyanosis, erythromelalgia, familial hemorrhage, histaminereaction, inflammatory papular and pustular lesions, lichen planus,lupus erythematosus, mycosis fungoides, neurodermatitis, neuropeptideand neurovascular reactions, parapsoriasis, perniosis (chilblains),photoallergy, photoreaction, photosensitivity, pityriasis rosea,pityriasis rubra pilaris, polymorphic light eruption, psoriasis,rhinophyma, rosacea, sclerosis, spider naevi, T-cell disorders,telangiectasia, varicose veins (varicosis), urticaria, vessel dilation,and other vascular reactions.

(E) Musculoskeletal System

The conditions or abnormalities of musculoskeletal system include, butare not limited to, the following conditions or disorders, which maypresent as indicated, or otherwise: (1) osteoporosis: reduction ofcalcium in bone leading to thin bone and bone susceptible to fracture;(2) osteoarthritis: inflammation of joint cartilage provoking swellingand pain; (3) rheumatoid arthritis: inflammation of synovium anddestruction of cartilage, damage to heart, lungs, nerves and eyes; (4)ankylosing spondylitis: arthritis affecting sacroiliac joints and spinewith inflammation and immovability; (5) bursitis: inflammation of bursa;(6) tendinitis: inflammation of tendon; (7) gout: recurrent acutearthritis from uric acid deposit; and (8) specifically, neck, shoulder,elbow, wrist, lower back, hip, knee and ankle pains, inflammation, andarthritis.

(F) Cutaneous System and Others

The cosmetic, dermatological or other conditions and disorders ofcutaneous system that can be treated with a composition of the presentinvention include, but are not limited to, infections, deranged ordisordered cutaneous or mucocutaneous tissue relevant to skin, nail andhair; oral, vaginal and anal mucosa; disturbed keratinization;inflammation; changes associated with intrinsic and extrinsic aging, andothers which may or may not be related to cutaneous system. Themanifestations include, but are not limited to, oily skin; acne;rosacea; age spots; blemished skin; blotches; cellulite; dermatoses;dermatitis; skin, nail and hair infections; dandruff; dryness orlooseness of skin, nail and hair; xerosis; inflammation, or eczema;elastosis; herpes; hyperkeratosis; hyperpigmented skin; ichthyosis;keratoses; lentigines; melasmas; mottled skin; pseudofolliculitisbarbae; photoaging and photodamage; pruritus; psoriasis; skin lines;stretch marks; thinning of skin, nail plate and hair; warts; wrinkles;oral or gum disease; irritated, inflamed, red, unhealthy, damaged orabnormal mucosa, skin, hair, nail, nostril, ear canal, anal or vaginalconditions; breakdown, defective synthesis or repair of dermalcomponents; abnormal or diminished synthesis of collagen,glycosaminoglycans, proteoglycans and elastin, as well as diminishedlevels of such components in the dermis; uneven skin tone; uneven andrough surface of skin, nail and hair; loss or reduction of skin, nailand hair resiliency, elasticity and recoilability; laxity; lack of skin,nail and hair lubricants and luster; fragility and splitting of nail andhair; yellowing skin; reactive, irritating or telangiectatic skin; anddull and older-looking skin, nail and hair. In addition, the compositionof the current invention can be used for general care of skin, nail andhair; to improve skin texture and pores, flakiness and redness; to makeskin soft, smooth, fresh, balanced, visibly clear, even-toned andbrighter; to increase skin fullness and plumpness; and for skin bleachand lightening and wound healing; to reduce or prevent sweating orperspiration of underarm, crotch, palm, or other parts of the body.

Skin, nail and hair infections can be caused by microorganisms whichinclude bacteria, fungi, yeasts, molds, parasites and viruses. Morespecifically, the bacterial infections can cause trichomycosisaxillaris, pitted keratolysis, erythrasma, impetigo, ecthyma,furunculosis (boils), carbuncle, scalded skin syndrome, toxic shocksyndrome, erysipelas, cellulitis, necrotizing fasciitis, erysipeloid,cat-scratch disease (Rochalimaea henselae), syphilis, lyme disease(Borrelia burgdorferi), cutaneous anthrax (Bacillus anthracis),gonococcal septicaemia, inoculation tuberculosis, scrofuloderma,tuberculides, erythema induratum, leprosy (Mycobacterium leprae),leishmaniasis and acute paronychia. The viral infections can cause viralwarts (human papilloma virus), varicella (chickenpox), herpes zoster(varicella-zoster), herpes simplex (herpesvirus hominis), molluscumcontagiosum, orf, AIDS (acquired immunodeficiency syndrome, humanimmunodeficiency virus, HIV), herpangina, mucocutaneous lymph nodesyndrome (Kawasaki's disease), Gianotti-Crosti syndrome (hepatitis Bvirus), measles, rubella and erythema infectiosum. The fungal infectionscan cause ringworm, tinea pedis (athlete's foot), tinea unguis (nailinfection), tinea hands, tinea groin, tinea trunk and limbs, tineacapitis (scalp), oral candidiasis, candida intertrigo, genitalcandidiasis, chronic paronychia, chronic mucocutaneous candidiasis,pityriasis versicolor, histoplasmosis, coccidioidomycosis,blastomycosis, sporotrichosis, actinomycosis and mycetoma (madura foot).

(G) Other Tissues or Systems

These conditions and diseases include vision disorders of eyes, vocaldesfunctions, gum and periodontal diseases, hearing loss, sexualdysfunctions, desired augmentation of breast and penis, and increasedbody strength. Aside from cataract and glaucoma, the vision disorderscan be due to near-sightedness (myopia) and far-sightedness (hyperopia).Enhanced strength of extrinsic and intrinsic eye muscles, along withincreased relaxation of eye nerves may help improve conditions of myopiaand hyperopia.

Weakness and poor quality of the voice can be caused by larynxdysfuction. Relaxation of laryngeal nerves and enhanced laryngeal musclemay help improve the quality and the strength of voice.

The preferred condition or disease to be treated according toembodiments of the present invention is selected from the groupconsisting of arthritis, cancer, immune, infections, inflammation,musculus, nerve, skin, and vasculature.

The more preferred condition or disease to be treated is selected fromthe group consisting of arthritis, Alzheimer's disease, aging relatedskin changes, age spots, breast cancer, cellulitis, dermatitis,dermatoses, dry skin, eczema, itch, infections, inflammation, jointdisorder, mottled skin, muscle disorder, pain, Parkinson's disease,photoaging, psoriasis, rosacea, stretch marks, varicose veins, viralinfections, wrinkles, for skin lightening, to enhance muscle strength,and to reduce or prevent sweating or perspiration of underarm, crotch,palm, or other parts of the body.

The most preferred condition or disease to be treated is selected fromthe group consisting of arthritis, aging related skin changes, agespots, cellulitis, dermatitis, dermatoses, eczema, itch, inflammation,joint disorder, mottled skin, rosacea, stretch marks, wrinkles, for skinlightening, and to reduce or prevent sweating or perspiration ofunderarm, crotch, palm or other parts of the body.

Physiological Functions, Pharmacological Actions and Therapeutic Effects

When a substance is found to modulate or normalize certain physiologicalfunctions, the resulted pharmacological actions can provide broadtherapeutic effects on related conditions, disorders, diseases, symptomsand syndromes; simply described as “related indications”. Therefore, therelated indications can be grouped into one single physiologicalfunction as follows.

(1) Disturbed Keratinization (DK).

Many skin disorders such as dry skin, ichthyosis, calluses, keratosisand acne (initiated by blackhead formation) are due to disturbedkeratinization (disturbed or abnormal skin formation). When a substanceis discovered to modulate or normalize keratinization, the substance isreasonably expected or predicted to improve those conditions ordisorders which are caused by a common cause of disturbedkeratinization.

Therefore, disturbed keratinization covers, but is not limited to dryskin; dryness or looseness of skin, nail and hair; xerosis; ichthyosis;calluses; keratoses; acne; rosacea; blemished skin; dandruff; unevenskin tone; uneven and rough surface of skin; abnormal skin texture andpores; flakiness and redness; and to improve or make skin soft, smooth,fresh, balanced, or visibly clear.

(2) Aging Related Changes of Skin, Nail and Hair (AG).

In “Rook's Textbook of Dermatology” by Burns et al. Wiley-Blackwell,2010, Vol. 1, page 8.22 under “The ageing skin”, it is described that“wrinkling of ageing skin is almost entirely the result of changes inthe dermis”. In fact, skin aging including wrinkles is due mainly toprogressive degeneration of dermal components; namely,glycosaminoglycans (GAGs), collagen and elastic fibers in the dermis. Ina publication by Ditre et al. J. Amer Acad Dermatol, 1996, pages187-195, under “Effects of α-hydroxy acids on photoaged skin: A pilotclinical, histologic, and ultrastructural study”, histologic andultrastructural studies show that skin plump or increased skin thicknesscaused by topical application of a substance results from a combinationof epidermal and dermal changes. In epidermal changes, the epidermisincreases in thickness, and the melanin pigmentation shows less clumpingof melanin resulting in lighter skin color and improved age spots. Indermal changes, there are increased amounts of both glycosaminoglycans(GAGs) and collagen fibers, and elastic fibers tend to be longer andthicker. Therefore, when a substance is found to plump or increase theskin thickness, the substance is reasonably expected or predicted toimprove aging related skin changes including fine lines, wrinkles,photoaging, age spots, blotches, hyperpigmented skin, mottled skin, andfor younger-looking skin and skin lightening.

In general, aging related skin changes covers, for example, fine lines;wrinkles; age spots; blotches; cellulite; elastosis; lentigine; mottledskin; photoaging and photodamage; stretch marks; thinning of skin, nailplate and hair; warts; wrinkles; breakdown, defective synthesis orrepair of dermal components; abnormal or diminished synthesis ofcollagen, glycosaminoglycans, proteoglycans and elastin, as well asdiminished levels of such components in the dermis; loss or reduction ofskin, nail and hair resiliency, elasticity and recoilability; laxity;lack of skin, nail and hair lubricants and luster; fragility andsplitting of nail and hair; yellowing skin; and dull and older-lookingskin, nail and hair, even-toned and brighter; to increase skin fullnessand plumpness.

(3) Deranged Immune Disorders and Inflammation (DI).

The deranged or disturbed immune disorders can cause inflammation, pain,itch, swelling, edema, dermatitis, eczema, psoriasis, dermatoses, jointdisorders, and arthritis. When a substance is found to modulate ornormalize activities of immune cells by reducing inflammation, thesubstance is reasonably expected or predicted to improve the relatedindications or disorders.

Therefore, the deranged immune disorders cover, for example,inflammatory disorders; inflammation, dermatitis, or eczema; psoriasis;dermatoses; painful joints; arthritis; infections; Type 1 diabetes;viral hepatitis; inflammatory bowel disease; allergic food reactions;nephritis; vasculitis; vitiligo; multiple sclerosis; HIV and AIDS.

(4) Tumors and Cancers (CA).

Most tumors and cancers are caused by unregulated proliferation of cellsdue to loss of normal controls, resulting in abnormal growth, lack ofdifferentiation, local tissue invasion, and often, metastasis. When asubstance is found to normalize the control of cell growth, thesubstance is reasonably expected or predicted to improve or eradicatemost types of tumors and cancers including skin tumors and cancers,breast cancer, lung carcinoma, liver cancer, pancreatic cancer, coloncancers, and brain tumors.

Therefore, tumors and cancers cover: adrenal cancer, anus cancer, braintumor and cancer, bladder cancer, breast cancer, cervix cancer, coloncancers, endometrium cancer, esophagus cancer, Kaposi sarcoma, kidneycancer, larynx cancer, leukemia, lymphoma, lung cancer, liver cancer,oral cavity cancer, ovarian cancer, prostate cancer, pancreatic cancer,rectum cancer, skin cancer, stomach cancer, testis cancer, thyroidcancer, and uterine cancer.

(5) Nerve Disorders (ND).

Nervous system is very complex initiating from the brain, andcontrolling almost all the body functions. Only the dead cells or deadtissues such as nails, hair and stratum corneum do not contain nervefibers. Loss or malfunction of nerve cells can result in various nervedisorders, symptoms and syndromes.

Therefore, nerve disorders cover: dementia, Alzheimer's disease:progressive loss of memory, carpal tunnel syndrome, weakness, pain,tingling, numbness, burning in palm and fingers, encephalitis, headache,migraine, meningitis, neuralgia, peripheral neuropathy, sciatica,Parkinson's disease, amnesia, Bell's palsy, epilepsy, multiplesclerosis, paralysis and headache.

In view of the present disclosure, standard procedures can be performedto evaluate the effect of the administration of a composition to asubject, thus allowing a skilled artisan to determine thetherapeutically effective amount of the compound.

The clinically observable beneficial effect can be a situation that,when a composition of the present invention is administered to a subjectafter symptoms to be treated are observable, the symptoms are preventedfrom further development or aggravation, or develop to a lesser degreethan without administration of the specified composition according toembodiments of the present invention. The clinically observablebeneficial effect can also be that, when a composition of the presentinvention is administered to a subject before symptoms to be treated areobservable, the symptoms are prevented from occurring or subsequentlyoccur to a lesser degree than without administration of the compositionof the present invention.

In one embodiment, a therapeutically effective amount of theN-acyldipeptide derivative w reduce a syndrome or a condition ofdiscomfort of the subject to be treated by at least about 20%, forexample, by at least about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or about 100%.

In another embodiment, a therapeutically effective amount of theN-acyldipeptide derivative will prevent a syndrome or a condition ofdiscomfort of the subject to be treated, or reduce the probability ofits onset by at least about 20%, for example, by at least about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100%.

Dosages and dosing frequency will be determined by a trained medicalprofessional depending on the activity of the compounds used, thecharacteristics of the particular topical formulation, and the identityand severity of the dermatologic disorder treated or prevented.

Administration Routes and General Preparations

Another general aspect of the present invention relates to a compositionfor systemic or topical administration to a subject, the compositioncomprising a therapeutically effective amount of an N-acyldipeptidederivative according to an embodiment of the present invention and apharmaceutically or cosmetically acceptable carrier. Compositionsaccording to embodiments of the present invention can be formulated inany manner suited for topical or systemic administration to a subject.

Compositions comprising a bioactive dipeptide derivative of the presentinvention can be administered to a subject in need by topicalapplication, systemic or other routes. The topical application includesskin, eye, mucous membranes of the conjunctiva, nasopharynx, oropharynx,vagina, urethra, rectum, and anus. The systemic administration includesoral (enteral) administration and parenteral injections. The parenteralinjections include intravenous injection or infusion, intra-arterialinjection, subcutaneous injection, intramuscular injection, andintra-articular injection. Other routes of administration includesublingual administration, under the tongue, from oral mucosa bypassingthe portal circulation, and pulmonary adsorption by inhaling andabsorbing through the respiratory tract.

For topical application, the composition comprising a bioactivedipeptide derivative of the present invention can be formulated assolution, gel, lotion, cream, oil-in-water emulsion, water-in-oilemulsion, ointment, shampoo, spray, stick, powder, mask, pads, mouthrinse or wash, vaginal gel or suppositories, rectal gel orsuppositories, urethral gel or suppositories or other form acceptablefor use on skin, nail, hair, oral mucosa, vaginal or anal mucosa, mouthor gums. The concentration of an active ingredient can be about 0.001%to about 99.9% by weight or volume (solution composition) of the totalcomposition, with preferred concentration of about 0.01% to about 30%,and with more preferred concentration of about 0.1% to about 10% byweight or by volume (solution composition) of the total composition.

A typical gel composition may be formulated by the addition of a gellingagent, such as chitosan, methyl cellulose, ethyl cellulose, polyvinylalcohol, poly quatemiums, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate toa solution comprising the N-acyldipeptide derivative. The preferredconcentration of the gelling agent may range from 0.1 to 4 percent byweight of the total composition. In the preparation of shampoo, theN-acyldipeptide derivative may be first dissolved in water or propyleneglycol, and the solution thus obtained may be mixed with a shampoo base.Concentrations of the N-acyldipeptide derivative used in gel or shampooform are the same as described above.

To prepare a solution composition, at least one bioactive dipeptidederivative of the present invention is dissolved in a solution preparedfrom water, ethanol, propylene glycol, butylene glycol, or othertopically acceptable solvent. To prepare a topical composition, abioactive dipeptide derivative of the present invention can beincorporated as a fine powder form without dissolving, or firstdissolving in water, ethanol, propylene glycol or other solvent, and thesolution thus obtained is mixed with a topically acceptable base orvehicle including gel, lotion, cream, oil-in-water emulsion,water-in-oil emulsion, ointment, shampoo, spray, stick, powder, mask,pads, mouth rinse or wash, vaginal gel or suppositories, and rectal gelor suppositories. Contemplated embodiments of the present inventioninclude concentration ranges of 0.001% to 0.01%, 0.01% to 0.1%, 0.1% to0.2%, 0.2% to 0.3%, 0.3% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 2%, 2% to 3%, 3% to4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to14%, 14% to 18%, 18% to 22%, 22% to 26%, 26% to 30%, 30% to 35%, 35% to40%, 40% to 45%, 45% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to90%, and 90% to 99.9% by weight or volume of the total composition.

The choice of topically administrable composition will depend on severalfactors, including the nature of the symptoms to be treated orprevented, the physiochemical characteristics of the particular compoundto be administered and of other excipients present, their stability inthe formulation, available manufacturing equipment, and costconstraints.

For systemic use or other routes of administration, a bioactivedipeptide derivative of the present invention can be formulated for oraladministration, parenteral injections or other routes including oralmucosa, under the tongue administration with or without pharmaceuticallyacceptable vehicle or carrier.

In oral preparations, a bioactive dipeptide derivative of the presentinvention is formulated in powder, tablet form, gelatin capsules with orwithout mixing with gelatin powder, or in other form including a liquidor suspension form. Each tablet, capsule or unit dosage contains about0.01 mg to about 100 mg, preferably about 0.1 mg to about 50 mg, andmore preferably about 1 mg to about 25 mg of the bioactive dipeptidederivative. As an illustration, bioactive dipeptide derivative powder, 1mg, can be placed under the tongue without swallowing for a short timeto achieve systemic administration. The daily dosage for a subject canvary, however in general is about 0.001 mg/kg to about 10 mg/kg,preferably about 0.01 mg to about 5 mg/kg, and more preferably about 0.1mg to about 2 mg/kg body weight of the subject.

For parenteral injections, a bioactive dipeptide derivative is preparedin a solution or suspension under sterilized conditions in concentrationfrom about 0.01% to about 10%, preferably about 0.1% to about 5%, morepreferably about 0.2% to about 2% weight by volume in water, propyleneglycol, glycerol, polyethylene glycol, a mixture thereof, or in othervehicle or carrier. The other vehicle or carrier includes peanut oil,soybean oil, mineral oil, sesame oil, and the like. As an option, athickener can be added into an injection composition to increase theviscosity, so that the composition has a comparable viscosity with thebody fluid in the knee joints or other joints. As an illustration, butnot limitation, the thickener can be selected from the group consistingof carboxymethylcellulose, sodium carboxymethylcellulose, casein,cellulose, gelatin, sodium hyaluronate, methylcellulose, PEG 200, PEG300, PEG 400, PEG 600, PEG 3350, PEG 4000, polyglactin, polylactide,polypropylene glycol, polyvinyl alcohol, protamine sulfate, povidone,starch, captisol, dextran, dextrose, fructose, albumin, and lactose.

In another embodiment, the composition may further comprise anadditional cosmetic, pharmaceutical, or other agent to achievesynergetic or synergistic effects. To prepare a topical combinationcomposition, a cosmetic, pharmaceutical or other agent is incorporatedinto any one of the above compositions by dissolving or mixing the agentinto the formulation. Other forms of compositions for delivery of theN-acyldipeptide derivative of the present invention are readilyrecognized by those skilled in the art.

A composition comprising the N-acyldipeptide derivative may be takenorally one to three times, preferably twice daily, for prevention ortreatment of disorders and diseases associated with immune, tumors,cancers, nervous, vascular, musculoskeletal, cutaneous system, or othertissues and systems. The oral administration may continue until thesymptom or disease has been eradicated or substantially improved. Thesymptoms or disorders include, for example, pains, pruritus,inflammation, erythema, dermatitis, acne, eczema, dementia, Alzheimer'sdisease, joint pain or swelling, and arthritis.

The bioactive dipeptide derivative of the present invention is believedto be therapeutically effective to alleviate or improve conditions,disorders, diseases, symptoms or syndromes associated with immune,nervous, vascular, musculoskeletal, cutaneous system, other tissues andsystems, or for regulation and treatment of abnormal cell growthincluding tumors and cancers. The composition containing a bioactivedipeptide derivative of the present invention can be administered aloneor in combination with another active agent. The composition and theother active agent can be administered simultaneously or sequentially.

Other forms of compositions for delivery of the compound of the presentinvention are readily blended, prepared or formulated by those skilledin the art.

A composition comprising a bioactive dipeptide derivative isadministered to a subject in various means that are acceptable for theconditions to be treated.

In one embodiment, the composition was topically applied to the skin.For example, a solution or cream containing 0.1% to 1% by weight ofN-acyldipeptide or N-acyldipeptide amide, such as N—Ac-L-Tyr-L-Tyr-NH₂,or N—Ac-L-Val-L-Ala-NH₂ was topically applied to an involved skin onceor twice daily for several weeks or until a desired therapeutic effecthad been achieved.

The composition can also be administered systemically or by otherroutes, such as via oral administration or parenteral injection. Forexample, N—Ac-L-Val-L-Ala-NH2 0.2% (w/w) in water, 1 ml (2 mg) wasinjected intra-articularily into a knee of a subject to relieve thearthritis pain and inflammation.

The composition can be administered alone or optionally in combinationwith another active ingredient. For example, a corticosteroid,hydrocortisone-17-valerate 0.2% (w/w) was incorporated into a topicalcomposition containing 0.5% (w/w) N—Ac-L-Val-L-Ala-NH2 to rapidlyimprove chronic eczema lesions. The composition and the other activeingredient can be administered simultaneously or sequentially. Undersuch cooperative actions, the N-acyldipeptide derivative and thecorticosteroid mutually provided synergetic, synergistic, or enhancingeffects for the anti-inflammatory actions.

For synergetic, synergistic, additive, enhancing, or other mutuallycooperative beneficial effects, a cosmetic, pharmaceutical, or otheragent can be incorporated into the composition of the present inventionor administered independently at the same time or different time. Theseagents include but are not limited to hydroxyacids, ketoacids andrelated compounds; phenyl alpha acyloxyalkanoic acids and derivatives;N-acyl-aldosamines, N-acylamino acids and related N-acyl compounds;local analgesics and anesthetics; anti-acne agents; anti-bacterialagents; anti-yeast agents; anti-fungal agents; anti-viral agents;anti-infective agents; anti-dandruff agents; anti-dermatitis agents;anti-eczema agents; anti-histamine agents; anti-pruritic agents;anti-emetics; anti-motion sickness agents; anti-inflammatory agents;anti-hyperkeratotic agents; antiperspirants; anti-psoriatic agents;anti-rosacea agents; anti-seborrheic agents; hair conditioners and hairtreatment agents; anti-aging and anti-wrinkle agents; anti-anxietyagents; anti-convulsant agents; anti-depressant agents; antineoplasticagents; sunblock and sunscreen agents; skin lightening agents;depigmenting agents; astringents; cleansing agents; corn, callus andwart removing agents; skin plumping agents; skin volumizing agents; skinfirming agents; matrix metalloproteinase (MMP) inhibitors; topicalcardiovascular agents; wound-healing agents; gum disease or oral careagents; amino acids; tripeptides; oligopeptides; polypeptides;carbohydrates; aminocarbohydrates; vitamins; corticosteroids; tanningagents; hormones; retinoids; peroxides; peracids; superoxides, ozonides,persulfates, and active agents.

The above agents include, but are not limited to, the following:abacavir, abciximab, abelcet, acamprosate, acarbose, acebutolol,acetaminophen, acetaminosalol, acetazolamide, acetic acid, aceticperacid, acetic peroxide, acetohydroxamic acid, acetylcysteine,acetylsalicylic acid, N-acylglutathione esters, acitretin, aclovate,acrivastine, acthrel, actidose, actigall, actiq, acyclovir, adalimumab,adapalene, adefovir dipivoxil, adenosine, agalsidase, albendazole,albumin, albuterol, alclometasone dipropionate, aldesleukin, alefacept,alemtuzumab, alendronate, alfuzosin, alitretinoin, allantoin, allium,allopurinol, alloxanthine, almotriptan, alosetron, alpha tocopheral,alphal-proteinase, alprazolam, alprenolol, alprostadil, alteplase,altretamine, aluminum acetate, aluminum chloride, aluminumchlorohydroxide, aluminum hydroxide, amantadine, amifostine, amiloride,aminacrine, amino acid, aminobenzoate, p-aminobenzoic acid, aminocaproicacid, aminohippurate, aminolevulinic acid, aminosalicylic acid,amiodarone, amitriptyline, amlodipine, amocarzine, amodiaquin,amorolfine, amoxapine, amoxicillin, amphetamine, amphotericin,ampicillin, amprenavir, anagrelide, anakinra, anastrozole, anisindione,anthralin, antihemophilic, antithrombin, anti-thymocyte, antivenin,apomorphine, aprepitant, aprotinin, arbutin, argatroban, aripiprazole,ascorbic acid, ascorbyl palmitate, aspirin, atazanavir, atenolol,atomoxetine, atorvastatin, atovaquone, atropine, azathioprine, azelaicacid, azelastine, azithromycin, baclofen, bacitracin, balsalazide,balsam, basiliximab, beclomethasone dipropionate, bemegride, benazepril,bendroflumethiazide, benzocaine, benzoic acid, benzonatate,benzophenone, benzoyl peroxide, benztropine, bepridil, beta carotene,betamethasone dipropionate, betamethasone valerate, betaxolol,bethanechol, bevacizumab, bexarotene, bicalutamide, bimatoprost,bioflavonoids, biotin, biperiden, bisacodyl, bisoprolol, bivalirudin,bortezomib, bosentan, botulinum, brimonidine, brinzolamide,bromocriptine, brompheniramine, budesonide, bumetanide, bupivacaine,buprenorphine, bupropion, burimamide, buspirone, busulfan, butabarbital,butalbital, butenafine, butoconazole, butorphanol, butyl aminobenzoate,cabergoline, caffeic acid, caffeine, calcipotriene, calcitonin-salmon,calcitriol, calcium peroxide, calfactant, Camellia sinensis, camphor,candesartan cilexetil, capecitabine, capreomycin, capsaicin, captopril,carbamazepine, carbamide peroxide, carbidopa, carbinoxamine, cefditorenpivoxil, cefepime, cefpodoxime proxetil, celecoxib, cetirizine,cevimeline, chitosan, chlordiazepoxide, chlorhexidine, chloroquine,chlorothiazide, chloroxylenol, chlorpheniramine, chlorpromazine,chlorpropamide, ciclopirox, cilostazol, cimetidine, cinacalcet,ciprofloxacin, citalopram, citric acid, cladribine, clarithromycin,clemastine, clindamycin, clioquinol, clobetasol propionate, clocortolonepivalate, clomiphene, clonidine, clopidogrel, clotrimazole, clozapine,coal tar, coal tar extracts (LCD), codeine, cromolyn, crotamiton,cyclizine, cyclobenzaprine, cycloserine, cytarabine, dacarbazine,dalfopristin, dapsone, daptomycin, daunorubicin, deferoxamine,dehydroepiandrosterone, delavirdine, desipramine, desloratadine,desmopressin, desoximetasone, dexamethasone, dexmedetomidine,dexmethylphenidate, dexrazoxane, dextroamphetamine, diazepam,diclofenac, dicyclomine, didanosine, dihydrocodeine, dihydromorphine,diltiazem, 6,8-dimercaptooctanoic acid (dihydrolipoic acid),diphenhydramine, diphenoxylate, dipyridamole, disopyramide, dobutamine,dofetilide, dolasetron, donepezil, dopa esters, dopamide, dopamine,dorzolamide, doxepin, doxorubicin, doxycycline, doxylamine, doxypin,duloxetine, dyclonine, econazole, efalizumab, eflornithine, eletriptan,emtricitabine, enalapril, ephedrine, epinephrine, epinine, epirubicin,eptifibatide, ergotamine, erythromycin, escitalopram, esmolol,esomeprazole, estazolam, estradiol, etanercept, ethacrynic acid, ethinylestradiol, etidocaine, etomidate, famciclovir, famotidine, felodipine,fentanyl, ferulic acid, fexofenadine, flecainide, fluconazole,flucytosine, fluocinolone acetonide, fluocinonide, 5-fluorouracil,fluoxetine, fluphenazine, flurazepam, fluticasone propionate,fluvoxamine, formoterol, furosemide, galactarolactone, galactonic acid,galactonolactone, galantamine, gatifloxacin, gefitinib, gemcitabine,gemifloxacin, glucarolactone, gluconic acid, gluconolactone, glucuronicacid, glucuronolactone, glutathione, glycolic acid, griseofulvin,guaifenesin, guanethidine, N-guanylhistamine, haloperidol, haloprogin,hexylresorcinol, homatropine, homosalate, hormone, hydralazine,hydrochlorothiazide, hydrocortisone, hydrocortisone 21-acetate,hydrocortisone 17-butyrate, hydrocortisone 17-valerate, hydrogenperoxide, hydromorphone, hydroquinone, hydroquinone monoether,hydroxyzine, hyoscyamine, hypoxanthine, ibuprofen, ichthammol,idarubicin, imatinib, imipramine, imiquimod, indinavir, indomethacin,infliximab, irbesartan, irinotecan, isoetharine, isoproterenol,itraconazole, kanamycin, ketamine, ketanserin, ketoconazole, ketoprofen,ketotifen, kojic acid, labetalol, lactic acid, lactobionic acid,lamivudine, lamotrigine, lansoprazole, letrozole, leuprolide,levalbuterol, levofloxacin, lidocaine, linezolid, lobeline, loratadine,loperamide, losartan, loxapine, lysergic diethylamide, mafenide, malicacid, maltobionic acid, mandelic acid, maprotiline, mebendazole,mecamylamine, meclizine, meclocycline, memantine, menthol, meperidine,mepivacaine, mequinol, mercaptopurine, mescaline, metanephrine,metaproterenol, metaraminol, metformin, methadone, methamphetamine,methotrexate, methoxamine, methyldopa esters, methyldopamide,3,4-methylenedioxymethamphetamine, methyllactic acid, methyl nicotinate,methylphenidate, methyl salicylate, metiamide, metolazone, metoprolol,metronidazole, mexiletine, miconazole, midazolam, midodrine, miglustat,minocycline, minoxidil, mirtazapine, mitoxantrone, moexiprilat,molindone, monobenzone, morphine, moxifloxacin, moxonidine, mupirocin,nadolol, naftifine, nalbuphine, nalmefene, naloxone, naproxen,nefazodone, nelfinavir, neomycin, nevirapine, niacin, niacinamide,nicardipine, nicotine, nifedipine, nimodipine, nisoldipine,nitrofurantoin, nizatidine, norepinephrine, nystatin, octopamine,octreotide, octyl methoxycinnamate, octyl salicylate, ofloxacin,olanzapine, olmesartan medoxomil, olopatadine, omeprazole, ondansetron,oxiconazole, oxotremorine, oxybenzone, oxybutynin, oxycodone,oxymetazoline, padimate O, palonosetron, pantothenic acid, pantoyllactone, paroxetine, pemoline, penciclovir, penicillamine, penicillins,pentazocine, pentobarbital, pentostatin, pentoxifylline, peptide,perazine, pergolide, perindopril, permethrin, phencyclidine, phenelzine,pheniramine, phenmetrazine, phenobarbital, phenol, phenoxybenzamine,phentolamine, phenylephrine, phenylpropanolamine, phenytoin,physostigmine, pilocarpine, pimecrolimus, pimozide, pindolol,pioglitazone, pipamazine, piperonyl butoxide, pirenzepine, podofilox,podophyllin, potassium peroxide, povidone iodine, pramipexole,pramoxine, prazosin, prednisone, prenalterol, prilocaine, procainamide,procaine, procarbazine, promazine, promethazine, promethazinepropionate, propafenone, propoxyphene, propranolol, propylthiouracil,protein, protriptyline, pseudoephedrine, pyrethrin, pyrilamine,pyrimethamine, quetiapine, quinapril, quinethazone, quinidine,quinupristin, rabeprazole, reserpine, resorcinol, retinal,13-cis-retinoic acid, retinoic acid, retinol, retinyl acetate, retinylpalmitate, ribavirin, ribonic acid, ribonolactone, rifampin,rifapentine, rifaximin, riluzole, rimantadine, risedronic acid,risperidone, ritodrine, rivastigmine, rizatriptan, ropinirole,ropivacaine, salicylamide, salicylic acid, salmeterol, scopolamine,selegiline, selenium sulfide, serotonin, sertaconazole, sertindole,sertraline, shale tar, sibutramine, sildenafil, sotalol, streptomycin,strychnine, sulconazole, sulfacetamide, sulfabenz, sulfabenzamide,sulfabromomethazine, sulfacetamide (sodium sulfacetamide),sulfachlorpyridazine, sulfacytine, sulfadiazine, sulfadimethoxine,sulfadoxine, sulfaguanole, sulfalene, sulfamethizole, sulfamethoxazole,sulfanilamide, sulfapyrazine, sulfapyridine, sulfasalazine,sulfasomizole, sulfathiazole, sulfisoxazole, sulfur, tacrolimus,tadalafil, tamsulosin, tartaric acid, tazarotene, tegaserol,telithromycin, telmisartan, temozolomide, tenofovir disoproxil,terazosin, terbinafine, terbutaline, terconazole, terfenadine,tetracaine, tetracycline, tetrahydrozoline, thalidomide, theobromine,theophylline, thiabendazole, thiethylperazine, thioctic acid (lipoicacid), thioridazine, thiothixene, thymol, tiagabine, timolol,tinidazole, tioconazole, tirofiban, tizanidine, tobramycin, tocainide,tolazoline, tolbutamide, tolnaftate, tolterodine, tramadol,tranylcypromine, trazodone, triamcinolone acetonide, triamcinolonediacetate, triamcinolone hexacetonide, triamterene, triazolam,triclosan, triflupromazine, trimethoprim, trimipramine, tripelennamine,triprolidine, tromethamine, tropic acid, tyramine, undecylenic acid,urea, urocanic acid, ursodiol, valacyclovir, vardenafil, venlafaxine,verapamil, vitamin, vitamin E acetate, voriconazole, warfarin, wood tar,xanthine, zafirlukast, zaleplon, zinc pyrithione, ziprasidone,zolmitriptan and zolpidem.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

Example 1

In one of the studies related to skin changes associated with aging,skin thickness was measured by micrometer calipers as follows.

The skin was grasped with a 2×6 cm metal hinge; the internal faces ofwhich were coated with emery cloth to prevent slippage, and manuallysqueezed to threshold subject discomfort. Combined thickness of twowhole-skin layers including thickness of the two hinge leaves wasmeasured with micrometer calipers. Thickness of the two hinge leaves wassubtracted to determine the actual thickness of two whole-skin layers.Triplicate measurements on treated sites were done and an average numberwas used for calculation of the skin thickness.

In other studies, test sites of skin 17 mm in diameter were used, thecircular sites were marked with permanent ink. Intervening control siteswere also 17 mm in diameter. Thickness of skin of all sites was measureddirectly by means of an electronic digital caliper. In this instance thejaws of the caliper were opened to 17 mm, applied with pressure to theskin sites and then closed to firm tightness. Thickness of skin was thenread off the screen of the calipers. Measurements of all sites were madein triplicates.

Example 2

A typical oil-in-water emulsion or cream composition containing abioactive dipeptide derivative of the present invention was formulatedas follows.

A bioactive dipeptide derivative of the present invention 0.2 g wasdissolved in 39.8 ml warm solution prepared from 80 parts water and 20parts propylene glycol by volume (hereinafter referred to as WP82). Thesolution or suspension thus obtained was mixed with 60 g oil-in-wateremulsion or cream. The oil-in-water emulsion or cream thus formulatedcontained 0.2% bioactive dipeptide derivative of the present inventionin oil-in-water emulsion or cream. Under similar conditions, emulsion orcream compositions containing 0.01% to 10% bioactive dipeptidederivative of the present invention were formulated.

As an illustration, N—Ac-L-Val-L-Ala-NH2 powder 0.2 g was dissolved in39.8 ml warm WP82 solution. The clear solution thus obtained was mixedwith 60 g oil-in-water emulsion or cream. The oil-in-water emulsion orcream thus formulated contained 0.2% N—Ac-L-Val-L-Ala-NH₂. Under thesimilar conditions, the following emulsion or cream compositions wereformulated:

N—Ac-L-Ile-L-Ala-NH₂ 1% (w/w) emulsion or cream;N—Ac-L-Leu-L-Ala-NH₂ 1% (w/w) and 5% (w/w) emulsions or creams.N—Ac-L-Val-L-Ala-NH₂ 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1% and2%, all by weight,emulsions or creams.

Vehicle control emulsion or cream composition was prepared in the samemanner except that the bioactive dipeptide derivative of the presentinvention was replaced by water.

Example 3

As another illustration to Example 2, 5 g N—Ac-L-Leu-Gly-NH2 powder wasdissolved in 35 ml warm WP82 solution. The clear solution thus obtainedwas mixed with 60 g oil-in-water emulsion or cream. The oil-in-wateremulsion or cream thus formulated contained 5% (w/w) N—Ac-L-Leu-Gly-NH2.

Under the similar conditions, the following emulsion or creamcompositions were formulated, all percentages are by weight:

N—Ac-β-Ala-Gly-NH₂ 1% in emulsion or creamN—Ac-L-Asn-Gly-NH₂ 4% in emulsion or creamN—Ac-Abz-Gly-NH₂ 5% in emulsion or creamN—Ac-L-Cys-Gly-NH₂ 10% in emulsion or creamN—Ac-Gaba-Gly-NH₂ 8% in emulsion or creamN—Ac-Gly-Gly-NH₂ 5% in emulsion or creamN—Ac-L-His-Gly-NH₂ 5% in emulsion or creamN—Ac-L-Ile-Gly-NH₂ 0.5%, 1%, 2%, 5%, 6%, and 10% in emulsions or creamsN—Ac-L-Leu-Gly-NH₂ 5%, 6%, 7%, 8% and 10% emulsions or creamsN—Ac-L-Pro-Gly-NH₂ 3% in emulsion or creamN—Ac-L-Ser-Gly-NH₂ 5% in emulsion or creamN—Z-L-Tyr-Gly-NH₂ 5% in emulsion or creamN—Ac-L-Val-Gly-NH₂ 5% and 10% in emulsions or creams

Example 4

As another illustration to Example 2, and under the similar conditions,the following emulsion or cream compositions were also formulated, allpercentages are by weight:

N—Ac-βAla-L-His-NH₂ 5% in emulsion or creamN,N′-diAc-βAla-His-NH₂ 5% in emulsion or creamN—Ac-βAla-L-His-OH 5% in emulsion or creamN—Ac-βAla-L-His-OEt 5% in emulsion or creamN—Ac-L-Ala-L-Ile-NH₂ 1% in emulsion or creamN—Ac-L-Ile-L-Ile-NH₂ 1% in emulsion or creamN—Ac-L-Leu-L-Leu-NH₂ 1% in emulsion or creamN—Ac-L-Ile-L-Tyr-NH₂ 1% in emulsion or creamN—Ac-L-Leu-L-Tyr-NH₂ 1% in emulsion or creamN—Ac-L-Tyr-L-Tyr-NH₂ 1% in emulsion or creamN—Ac-L-Val-L-Tyr-NH₂ 5% in emulsion or creamN—Ac-L-Tyr-L-Tyr-OH 1% in emulsion or creamN—Ac-L-Tyr-L-Tyr-OEt 1% in emulsion or creamN—Ac-L-Tyr-L-Tyr-NHNH₂ 0.6% in emulsion or creamN—Ac-L-Tyr-L-Tyr-NHNHAc 0.7% in emulsion or cream

Example 5

A typical solution composition containing a bioactive dipeptidederivative of the present invention was formulated as follows.

A bioactive dipeptide derivative of the present invention, 0.1 g, wasdissolved in 99.9 ml solution prepared from 40 parts water, 40 partsethanol and 20 parts propylene glycol by volume (hereinafter referred toas WEP442). The solution thus formulated contained 0.1% (w/w) bioactivedipeptide derivative of the present invention in solution composition.Under similar conditions, solution compositions containing 0.01% to 10%,by weight, bioactive dipeptide derivative of the present invention wereformulated.

As an illustration, N—Ac-L-Val-L-Ala-NH2 powder, 0.1 g, was dissolved in99.9 ml WEP442. The solution composition thus formulated had pH 5.4, andcontained 0.1% (w/w) N—Ac-L-Val-L-Ala-NH2.

Under the similar conditions, solution compositions were formulated asfollows, wherein all percentages are by weight:

N—Ac-L-Val-L-Ala-NH₂: 0.01%, pH 4.7; 0.05%, pH 5.0; 0.1%, pH 5.4; 0.2%,pH 5.6; 0.4%, pH 5.8; 0.5%, pH 5.9; all in WEP442 N—Ac-L-Ile-L-Ala-OH:0.4% in WEP442, pH 3.9 N—Ac-L-Leu-L-Ala-OH: 0.4% in WEP442N—Ac-L-Val-L-Ala-OH: 0.4% in WEP442, pH 3.0 N—Pr-L-Val-L-Ala-OH: 0.4% inWEP442, pH 3.7 N—Ac-L-Cys-L-Cys-NH₂: 0.4% in WEP442 N—Ac-L-Cys-L-Cys-OH:0.5% in WEP442, pH 5.3 N—Ac-L-Ile-Gly-OH: 0.4% in WEP442, pH 3.2N—Ac-L-Cys-Gly-NH₂: 1% in WEP442, pH 6.4 N—Ac-L-Leu-Gly-NH₂: 1%, pH 5.3;2.5%, pH 5.4; 5%, pH 5.4; 10% pH 5.4; all in WEP442. N—Ac-L-Leu-Gly-OH:0.4% in WEP442, pH 3.8 N—Pr-L-Leu-Gly-OH: 0.4% in WEP442, pH 3.5

N—Ac-L-Pro-Gly-OH: 5% in 90 parts water and 10 parts propylene glycol byvolume (WP91), pH 6.6

N—Ac-L-Tyr-Gly-NH₂: 2.5% in WEP442 N—Ac-L-Gln-Gly-NH₂: 2.5% in WEP442N—Ac-L-Val-Gly-OH: 0.4% in WEP442, pH 3.2 N—Ac-Gly-L-Pro-NH₂: 5% inWEP442 βAla-L-His-NH₂: 5% in WEP442, pH 6.5 N,N′-DiAc-βAla-L-His-NH₂: 3%in WEP442, pH 7.0; 4% in WP82, pH 7.1 N—Ac-Gaba-Gly-NH₂: 5% in WEP442,pH 5.8 N—Ac-βAla-Gly-NH₂: 5% in WEP442, pH 5.2 N—Ac-Gly-L-Pro-NH₂: 5% inWEP442 N—Ac-L-Cys-L-Tyr-NH₂: 1% in WEP442, pH 6.4

N—Ac-L-Tyr-L-Tyr-OEt: 1% in a solution prepared from 80 parts ethanoland 20 parts propylene glycol by volume (hereinafter referred to asEP82)

N—Ac-L-Tyr-L-Tyr-NH₂: 0.5%, 1% in EP82; 0.5%, 3% in WEP442, pH 6.7N—Ac-L-Tyr-L-Tyr-OH: 0.3%, 0.4%, 0.5%, or 1% in WEP442, all pH 3.7N—Ac-L-Tyr-L-Tyr-NHNH₂: 0.3% in WEP442, pH 5.8; 2% in WEP442N—Ac-L-Tyr-L-Tyr-NHNHAc: 0.3% and 0.5% in WEP442, pH 5.5; 3% in WEP442N—Ac-L-Phe-L-Phe-NH₂: 0.4% in WEP442 N—Ac-L-Trp-L-Trp-NH₂: 0.4% inWEP442

Vehicle control compositions such as WEP442 and EP82 were prepared inthe same manner but without any bioactive dipeptide derivative of thepresent invention.

Example 6

A female subject, age 41, topically applied twice dailyN—Ac-L-Tyr-L-Tyr-NH2 3% (w/v) in WEP442, pH 6.7 as prepared in Example5, to her left forearm, and a vehicle control WEP442 to her rightforearm for three weeks. After 3 weeks, while there was no change inskin thickness of her right forearm, her left forearm had increased 14%in skin thickness as measured by the micrometer calipers as shown inExample 1. After 4 weeks her vehicle treated right forearm was stillloose, relatively thin and wrinkled when lifted. In contrast, her leftforearm was more firm, smooth, plump, and less wrinkled when lifted.While there was no change in skin thickness of her right forearm, herleft forearm had increased 17% in skin thickness as measured by themicrometer calipers. This result indicates that N—Ac-L-Tyr-L-Tyr-NH₂ hasthe potential of providing therapeutic effects for topical treatment ofdisturbed keratinization and aging related changes of skin, nail andhair.

Example 7

A female subject, age 52, topically applied twice dailyN—Ac-L-Tyr-L-Tyr-NH2 3% (w/v) in WEP442, pH 6.7 as prepared in Example5, to her left forearm, and a vehicle control WEP442 to her rightforearm for two weeks. After two weeks her vehicle treated right forearmwas still loose, relatively thin and wrinkled when lifted. In contrast,her left forearm was more firm, smooth, plump, and less wrinkled whenlifted. While there was no change in skin thickness of her rightforearm, her left forearm had increased 13% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. This resultshows that N—Ac-L-Tyr-L-Tyr-NH2 has the potential of providingtherapeutic effects for topical treatment of disturbed keratinizationand aging related changes of skin, nail and hair.

Example 8

A female subject, age 51, topically applied twice daily 2% (w/v)N—Ac-L-Tyr-L-Tyr-NHNH₂ in WEP442 as prepared in Example 5, to her leftforearm, and a vehicle control WEP442 to her right forearm for threeweeks. After one week while there was no change in skin thickness of herright forearm, her left forearm had increased 15% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. After threeweeks her vehicle treated right forearm was still loose, relatively thinand wrinkled when lifted. In contrast, her left forearm was more firm,smooth, plump, and less wrinkled when lifted. While there was no changein skin thickness of her right forearm, her left forearm had increased32% in skin thickness as measured by the micrometer calipers. Thisresult indicates that N—Ac-L-Tyr-L-Tyr-NHNH₂ has the potential ofproviding therapeutic effects for topical treatment of disturbedkeratinization and aging related changes of skin, nail and hair.

Example 9

A female subject, age 52, topically applied twice daily 2% (w/v)N—Ac-L-Tyr-L-Tyr-NHNH2 in WEP442 as prepared in Example 5, to her leftforearm, and a vehicle control WEP442 to her right forearm for twoweeks. After two weeks her vehicle treated right forearm was stillloose, relatively thin and wrinkled when lifted. In contrast, her leftforearm was more firm, smooth, plump, and less wrinkled when lifted.While there was no change in skin thickness of her right forearm, herleft forearm had increased 14% in skin thickness as measured by themicrometer calipers as shown in Example 1. This result indicates thatN—Ac-L-Tyr-L-Tyr-NHNH₂ has the potential for topical treatment ofdisturbed keratinization and aging related changes of skin, nail andhair.

Example 10

A female subject, age 51, topically applied twice daily 3% (w/v)N—Ac-L-Tyr-L-Tyr-NHNHAc in WEP442 as prepared in Example 5, to her leftforearm, and a vehicle control WEP442 to her right forearm for threeweeks. After one week while there was no change in skin thickness of herright forearm, her left forearm had increased 21% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. After threeweeks her vehicle treated right forearm was still loose, relatively thinand wrinkled when lifted. In contrast, her left forearm was more firm,smooth, plump, and less wrinkled when lifted. While there was no changein skin thickness of her right forearm, her left forearm had increased32% in skin thickness as measured by the micrometer calipers. Thisresult shows that N—Ac-L-Tyr-L-Tyr-NHNHAc has the potential of providingtherapeutic effects for topical treatment of disturbed keratinizationand aging related changes of skin, nail and hair.

Example 11

A female subject, age 73, topically applied twice daily 3% (w/v)N—Ac-L-Tyr-L-Tyr-NH2 in WEP442 as prepared in Example 5, to her leftforearm, and a vehicle control WEP442 to her right forearm for 4 weeks.After 2 weeks, there was no change in skin thickness of her rightforearm, her left forearm had increased 19% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. At the end of4 weeks, her vehicle treated right forearm was still loose, relativelythin and wrinkled when lifted. In contrast, her left forearm was morefirm, smooth, plump, and less wrinkled when lifted. While there was nochange in skin thickness of her right forearm, her left forearm hadincreased 21% in skin thickness as measured by the micrometer calipers.This result indicates that N—Ac-L-Tyr-L-Tyr-NH2 has the potential ofproviding therapeutic effects for topical treatment of disturbedkeratinization and aging related changes of skin, nail and hair.

Example 12

A male subject, age 79, having intensive itch and erythema on his leftarm from insect bites, topically applied 10% (w/w) N—Ac-L-Leu-Gly-NH₂cream as formulated in Example 3 on the involved lesions. The itchstopped within a few minutes, and the pruritus did not return for thenext 6 hours. The erythema and inflammation also improved after 12hours. The result shows that N—Ac-L-Leu-Gly-NH₂ has the potential ofproviding therapeutic effects for topical treatment of symptoms orsyndromes associated with nerve disorders and inflammation.

Example 13

A male subject, age 80, having chronic inflammation, erythema, eczemawith thick scales and itch on his right leg for more than 10 years,failed to respond with conventional treatments including topicalcorticosteroids. The involved skin was divided into two lesions fortesting. The subject topically applied twice daily to one lesion 0.5%(w/v) N—Ac-L-Tyr-L-Tyr-NH₂ solution composition, pH 6.7 prepared from 4parts water, 76 parts ethanol and 20 parts propylene glycol by volume,and to second lesion 0.4% (w/v) N—Ac-L-Val-L-Ala-NH₂ in WEP442 solutioncomposition, pH 5.8 as shown in Example 5. The itch stopped within a fewminutes, and the thick scales of both lesions started to disappear inthe next few days. At the end of 4 weeks, the erythema and thick scalesof both lesions disappeared almost completely and the treated skin siteswere smooth, soft, even-toned, brighter and more elastic when the skinwas stretched. The treated skin appeared much lighter in skin color ascompared to the surrounding untreated skin site. The treated skin had95-100% improvement as judged by clinical evaluation. The result showsthat both N—Ac-L-Tyr-L-Tyr-NH₂ and N—Ac-L-Val-L-Ala-NH₂ have thepotential of providing therapeutic effects for topical treatment ofsymptoms or syndromes associated with nerve disorders, disturbedkeratinization, aging skin, wrinkles, age spots, hyperpigmentation,inflammation, deranged immune system, and for skin lightening.

Example 14

A female subject, age 87, having chronic inflammation, dermatitis, oreczema with thick scales and itch on her right leg for more than 7years, failed to respond with conventional treatments including topicalcorticosteroids. The subject topically applied twice daily 10% (w/w)N—Ac-L-Leu-Gly-NH2 cream as formulated in Example 3 on the involvedlesions. The itch stopped within a few minutes, and the thick scalesstarted to disappear in the next few days. At the end of one week, thetreated skin had 50% improvement as judged by clinical evaluation. Theresult shows that N—Ac-L-Leu-Gly-NH2 has the potential of providingtherapeutic effects for topical treatment of symptoms or syndromesassociated with nerve disorders, disturbed keratinization, inflammationand deranged immune system.

Example 15

A female subject, age 50, having chronic inflammation, dermatitis, oreczema with scales, erythema and itch for more than 15 years, failed torespond with conventional treatments including topical corticosteroids.The subject topically applied twice daily 7% (w/w) N—Ac-L-Leu-Gly-NH₂oil-in-water emulsion as formulated in Example 3 on the involvedlesions. The itch stopped within a few minutes, and the lesions improvedfor the next few days. At the end of 10 days, the scales and erythemadisappeared almost completely and the treated skin had 90-100%improvement as judged by clinical evaluation. The result shows thatN—Ac-L-Leu-Gly-NH₂ has the potential for topical treatment of symptomsor syndromes associated with nerve disorders, disturbed keratinization,inflammation and deranged immune system.

Example 16

A female subject, age 43, having chronic inflammation, dermatitis, oreczema with scales, erythema and itch covering large areas of the bodyfor many years, failed to respond with conventional treatments includingtopical corticosteroids. The subject topically applied twice daily onthe involved lesions 6% (w/w) N—Ac-L-Leu-Gly-NH2 oil-in-water emulsionas formulated in Example 3. The itch stopped within a few minutes, andthe lesions improved for the next few days. At the end of 10 days, thescales and erythema disappeared almost completely and the treated skinhad 95% improvement as judged by clinical evaluation. The result showsthat N—Ac-L-Leu-Gly-NH2 has the potential for topical treatment ofsymptoms or syndromes associated with nerve disorders, disturbedkeratinization, inflammation and deranged immune system.

Example 17

A male subject, age 80, having chronic inflammation and eczema withthick scales and itch on his legs, failed to respond with conventionaltreatments including topical corticosteroids. The subject topicallyapplied twice daily to one leg 2% (w/v) N—Ac-L-Val-L-Ala-NH₂ cream asformulated in Example 2, and to another leg 0.5% (w/w)N—Ac-L-Tyr-L-Tyr-NHNHAc in WEP442, pH 5.5 as formulated in Example 5, onthe involved lesions. For both treated legs, the itch stopped within afew minutes, and the thick scales started to disappear in the next fewdays. The eczema lesions also improved substantially over the next fewweeks, and the skin became soft and smooth. At the end of four weeks,the treated legs had 95-100% improvement as judged by clinicalevaluations. The results show that the dipeptide derivatives of thepresent invention have the potential for topical treatment of symptomsor syndromes associated with nerve disorders, disturbed keratinization,inflammation and deranged immune system.

Example 18

A male subject, age 79, developed skin allergy from eating shellfishwith intensive itch and reddish papules on his back and thighs thatfailed to respond with conventional treatments including topicalcorticosteroids. The subject topically applied twice daily 0.5% (w/w)N—Ac-L-Val-L-Ala-NH₂ cream as formulated in Example 2 on the involvedlesions. The itch stopped within a few minutes, and the reddish papulesstarted to disappear in the next few days. At the end of 5 days, thetreated skin had 75% improvement as judged by clinical evaluation. Theresult shows that N—Ac-L-Val-L-Ala-NH₂ has the potential for topicaltreatment of symptoms or syndromes associated with nerve disorders,inflammation and deranged immune system.

Example 19

A male subject, age 80, having inflammatory dermatitis on both arms witherythema and intensive itch and failed to respond with conventionaltreatments including topical corticosteroids. The subject topicallyapplied twice daily 0.4% (w/v) N—Ac-L-Tyr-L-Tyr-OH in WEP442, pH 3.7 asformulated in Example 5 on the involved lesions. The itch stopped withina few minutes, and the erythema started to disappear in the next fewdays. At the end of one week, the treated skin had 95% improvement asjudged by clinical evaluation. The result shows that N—Ac-L-Tyr-L-Tyr-OHhas the potential for topical treatment of symptoms or syndromesassociated with nerve disorders and inflammation.

Example 20

A male subject, age 80, having chronic eczema with intensive itch,failed to respond with conventional treatments including topicalcorticosteroids. The subject took 1 mg N—Ac-L-Tyr-L-Tyr-OH powder underthe tongue until it was dissolved completely. The same dose was takentwice daily for the next three days. Systemic administration ofN—Ac-L-Tyr-L-Tyr-OH under the tongue did not provoke any irritation orside reactions. Such systemic administration of the bioactive dipeptidederivative of the present invention is believed to have the potential oftreating diseases, symptoms or syndromes associated with immune, tumors,cancers, nervous, vascular, musculoskeletal, cutaneous system, or othertissues and systems.

Example 21

A typical in vitro screen for anti-inflammatory effects was carried outas follows.

THP-1 leukemia cells (monocytic leukemic human cell line) were culturedand either untreated or treated with a bioactive dipeptide derivative(100 μg/ml) of the present invention for 24 hours. The untreated ortreated leukemia cells were then treated with angiocidin (10 μg/ml) for24 hours to induce interleukin-6 (IL-6) which was an indicator of theinflammation. The induced IL-6 was measured quantitatively by ELISA(enzyme-linked immunosorbent assay). The test results showed thatN—Ac-L-Val-L-Ala-NH2 inhibited the production of IL-6 by approximately70%. Therefore, the bioactive dipeptide derivatives of the presentinvention, such as N—Ac-L-Val-L-Ala-NH2, have the potential use asnonsteroidal anti-inflammatory substances.

Example 22

In one embodiment, a composition containing a bioactive dipeptidederivative of the present invention for systemic administration can beprepared as follows.

For injection administration, a composition can be prepared with orwithout a thickening agent, such as methyl cellulose. Methyl cellulose1% (w/v) in water solution was prepared by adding 1 g methyl cellulosein 90 ml water, and the mixture was gently homogenized. More water wasadded to make the final volume of 100 ml. The vehicle composition thusprepared contained 1% (w/v) methyl cellulose as a thickener.N—Ac-L-Tyr-L-Tyr-NH2, 100 mg, was dissolved in 5 ml water with orwithout 1% (w/v) methyl cellulose, and the solutions in injection vialswere sterilized at 1000C for 30 minutes. The compositions thus obtainedcontained 2% (w/v) or 20 mg/ml N—Ac-L-Tyr-L-Tyr-NH2 suitable forintra-articular, intralesional, or subcutaneous injection, or othersystemic administration.

Under the same conditions, the following compositions for systemicadministration were prepared, all percentages are by weight: 2% or 20mg/ml N—Ac-L-Tyr-L-Tyr-NHNH2 in water with or without 1% methylcellulose; 2% or 20 mg/ml N—Ac-L-Tyr-L-Tyr-NHNHAc in water with orwithout 1% methyl cellulose; 0.2% or 2 mg/ml N—Ac-L-Val-L-Ala-NH2 inwater with or without 1% methyl cellulose; and 0.2% or 2 mg/mlN—Ac-L-Tyr-L-Tyr-OH in water with or without 1% methyl cellulose.

Example 23

Bioactive dipeptide derivative of the present invention as potentialtreatment of knee osteoarthritis by intra-articular injections.

A male subject, age 90, had severe osteoarthritis of both knees for fourand half years. Prior therapy had included intra-articular injections ofcorticosteroids and hyaluronic acid as well as celecoxib (Celebrex)orally (200 mg) twice daily. Such therapy had provided only mildtransitory relief of knee pain and edema, and edema of lower legs.

Intra-articular injections of 0.2% (w/v) N—Ac-L-Val-L-Ala-NH2 in water,1 ml (2 mg) as prepared in Example 22 were administered to each knee.The pains in both knees disappeared 10-15 minutes after the injections,and the relief of pains lasted for 24 hours. Edema and inflammation ofthe knees and lower legs had diminished for approximately the same 24hour period. Repeat injections of the same composition were administeredonce a week for five weeks to provide continued relief of pain, edemaand inflammation.

In another trial, intra-articular injections of 0.2% (w/v)N—Ac-L-Val-L-Ala-NH2 with 1% (w/v) methyl cellulose in water, 0.4 ml(0.8 mg) as prepared in Example 22 were also administered to each kneeat different times. The pains in both knees disappeared 10-15 minutesafter the injections, and the relief of pains lasted for 24 hours. Edemaand inflammation of the knees and lower legs had diminished forapproximately the same 24 hour period.

The above results show that N-acyldipeptide derivatives of the presentinvention have the potential of treating inflammation, arthritis, pain,other immune and nerve disorders via systemic administration.

Example 24

A typical combination composition for synergetic, synergistic oradditive effects was prepared as follows.

N—Ac-L-Val-L-Ala-NH2 400 mg and N-acetyl-D-glucosamine (NAG) 400 mg weredissolved in 90 ml solution prepared from water 40 parts, ethanol 40parts and propylene glycol 20 parts by volume (WEP442), and the finalvolume was made up to 100 ml. The composition thus prepared had pH 5.6,and contained 0.4% (w/v) N—Ac-L-Val-L-Ala-NH2 and 0.4% (w/v) NAG.

A female subject, age 41, had inflammatory lesions on her left arm withintensive itch and reddish papules that failed to respond withconventional treatments including topical corticosteroids. The subjecttopically applied twice daily the above combination compositioncontaining 0.4% N—Ac-L-Val-L-Ala-NH2 and 0.4% NAG on the involvedlesions. The itch stopped within a few minutes, and the reddish papulesstarted to disappear in the next few days. At the end of 5 days, thetreated skin had more than 90% improvement as judged by clinicalevaluation. The treated skin sites showed that the skin was smooth andsoft to touch and appearance. The result shows that the combination ofN—Ac-L-Val-L-Ala-NH2 and NAG has synergetic or synergistic effects, andcan improve the lesions faster than the non-combination composition. Itis believed that the combination composition has the potential fortopical treatment of symptoms or syndromes associated with disturbedkeratinization, nerve disorders, inflammation and deranged immunesystem.

Example 25

As an another illustration, N—Ac-L-Tyr-L-Tyr-NH2, 500 mg, andhydrocortisone-17-valerate, 200 mg, were dissolved in 90 ml solutionprepared from 80 parts ethanol and 20 parts propylene glycol by volume(EP82), and the final volume was made up to 100 ml. The composition thusprepared contained 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH₂ and 0.2% (w/v)hydrocortisone-17-valerate.

A male subject, age 80, had chronic eczema on his right leg withintensive itch and thick scales, which failed to respond withconventional treatments including topical corticosteroids. The subjecttopically applied twice daily the above combination compositioncontaining 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH2 and 0.2% (w/v)hydrocortisone-17-valerate on the involved lesions. The itch stoppedwithin a few minutes, and the thick scales started to disappear in thenext few days. At the end of 7 days, the treated skin had 80%improvement as judged by clinical evaluation. The treated skin sitesshowed that the skin was smooth and soft to the touch and appearance.The result shows that the combination of N—Ac-L-Tyr-L-Tyr-NH2 andhydrocortisone-17-valerate has synergetic or synergistic effects, andcan improve the lesions much faster than the non-combinationcomposition. Thus, the combination composition has the potential fortopical treatment of symptoms or syndromes associated with disturbedkeratinization, nerve disorders, inflammation and deranged immunesystem.

Example 26

A male subject, age 41, had inflammatory eczema on his left hand withitch, erythema and some scales for 6 months, which failed to respondwith conventional treatments including topical corticosteroids. Thesubject topically applied twice daily the combination compositioncontaining 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH2 and 0.2% (w/v)hydrocortisone-17-valerate as described in Example 25 on the involvedlesions. The itch stopped within a few minutes, and the erythema andscales started to disappear in the next few days. At the end of 5 days,the treated skin had 75% improvement as judged by clinical evaluation.The treated skin sites showed that the skin was smooth and soft to thetouch and appearance. The result shows that the combination ofN—Ac-L-Tyr-L-Tyr-NH2 and hydrocortisone-17-valerate has synergetic orsynergistic effects, and can improve the lesions much faster than thenon-combination composition. Thus, the combination composition has thepotential for topical treatment of symptoms or syndromes associated withdisturbed keratinization, nerve disorders, inflammation and derangedimmune system.

Example 27

A typical example of a topical administration for systemic therapeuticeffects was carried out as follows. A male subject, age 80, havingsprained his left leg near the knee with inflammation for several weekshad severe pain each time walking. Massage therapy and conventionaltreatments provided only a temporary relief. The subject topicallyapplied and rubbed 0.4% (w/v) N—Ac-L-Val-L-Ala-NH2 in WEP442, pH 5.8 asprepared in Example 5 onto the left thigh and calf sites of skin for 30seconds at night. The next morning, the subject was surprised todiscover that he could walk slowly without feeling any pain on his leftleg. The inflammation in his left leg also subsided significantly. Theclinical evaluation showed that he had approximately 75% improvement onhis left leg.

The above result shows that topical administration of a bioactivedipeptide derivative of the present invention has the potential ofproviding therapeutic effects for pain, inflammation and arthritis.

Example 28

A male subject, age 80, had a skin cut with an open wound about 2 cmlong and a feeling of moderate pain on his left leg. The subjecttopically applied twice daily 0.5% (w/w) N—Ac-L-Val-L-Ala-NH2 cream asformulated in Example 2 on the wound. The pain stopped within a fewminutes, and the wound started to heal in the next few days. At the endof 5 days, the wound had healed nearly completely, and treated skinappeared normal without any signs of scar formation. The wound had95-100% improvement as judged by clinical evaluation. The result showsthat N—Ac-L-Val-L-Ala-NH2 has the potential for topical treatment ofskin wounds.

Example 29

Stretch marks, known as striae, are visible linear scars caused bychanges in collagen fibers and over stretching of the skin. Stretchmarks are quite common in most adult women, and they readily develop atpuberty or during pregnancy. They are also very common in obese peoplewith sudden loss of weight. At present, there is no cure or effectivetreatment.

A male subject, age 41, having lost a total of about 60 pounds fromoverweight over the last 10 years, developed multiple lesions of stretchmarks over the body. The subject selected two separate lesions ofstretch marks, and topically applied twice daily 0.5% (w/w)N—Ac-L-Val-L-Ala-NH2 cream (Example 2) on one lesion and 0.5% (w/v)N—Ac-L-Tyr-L-Tyr-NH2 solution in WEP442, pH 6.7 (Example 5) on the otherlesion with untreated lesions of stretch marks as a control.

In contrast to untreated lesions, the treated lesions of stretch marksslowly improved over the next few weeks. At the end of four weeks, thefine lines of stretch marks started to disappear, and deeper linesbecame less deep. The treated lesions had approximately 25% improvementas judged by clinical evaluation.

The above results show that a bioactive dipeptide derivative of thepresent invention has the potential of providing therapeutic effects viatopical administration to treat stretch marks, skin atrophy, skin scars,skin thinning, and other skin defects caused by changes to dermalcomponents including collagen and elastic fibers.

Example 30

A male subject, age 42, had multiple lesions of insect bites(mosquitoes) over the face and arms with itch and inflammatory lesionsof more than 1 cm in size. For testing purposes, the subject divided thelesions into three groups: the first group was treated with vehiclecontrol, the second and third groups were treated with activecompositions. The subject topically applied twice daily 0.5% (w/v)N—Ac-L-Val-L-Ala-NH₂ solution in WEP442, pH 5.9 on the lesions of thesecond group, and 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH₂ solution in WEP442, pH6.7 (Example 5) on the lesions of the third group. While there was noimmediate effect in the lesions treated with vehicle control in thefirst group, the itch stopped completely within a few minutes of topicalapplications in the second and third groups. The lesions in the secondand third groups also started to improve over the next few days. At theend of three days, while there was no discernible change in the lesionstreated with vehicle control in the first group, the lesions in thesecond and third groups had 90-100% improvement with nearly completedisappearance of inflammatory lesions.

The above results show that bioactive dipeptide derivatives of thepresent invention have the potential of providing therapeutic effectsvia topical administration to treat insect bites, inflammation, pain,other immune and nerve disorders.

Example 31

A male subject, age 42, having a sprained right shoulder withinflammation and pain caused by heavy lifting 6 weeks earlier did notrespond to conventional treatments. The subject topically applied twicedaily 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH2 in WEP442 solution, pH 6.7 asprepared in Example 5. After three days of topical application, the paindisappeared completely and the inflammation diminished substantially. Atthe end of 5 days, the right shoulder had 90% improvement as determinedby clinical evaluation. The result shows that the bioactive dipeptidederivative of the present invention has the potential for treating painand inflammation of joints by topical administration.

Example 32

A male subject, age 41, having muscle pain in both arms withinflammation caused by heavy lifting several weeks earlier did notimprove with the conventional treatments. The subject topically appliedtwice daily 0.5% (w/v) N—Ac-L-Val-L-Ala-NH2 in WEP442 solution, pH 6.7as prepared in Example 5 to the affected area of skin. The paindiminished with less inflammation over the next day. At the end of fivedays, the pain and inflammation disappeared completely, and the arms had95%-100% improvement as determined by clinical evaluation.

The result shows that the bioactive dipeptide derivative of the presentinvention has the potential of treating pain and inflammation of musclesby topical administration.

Example 33

A female subject, age 52, had multiple lesions of chigger bites (grassmites) over the legs with intensive itch and red inflammatory lesions.The subject topically applied twice daily 0.5% (w/v)N—Ac-L-Val-L-Ala-NH₂ solution in WEP442, pH 6.7 (Example 5) on thelesions. The itch stopped within a few minutes after the topicalapplication, and the red inflammatory lesions started to clear over thenext few days. At the end of five days, all red and inflammatory lesionsdisappeared completely, and the treated skin appeared normal. Thelesions on the legs had 100% improvement as judged by clinicalevaluation.

The above result shows that bioactive dipeptide derivatives of thepresent invention have the potential of providing therapeutic effectsvia topical administration to treat insect bites, inflammation, pain,other immune and nerve disorders.

Example 34

A female subject, age 73, topically applied twice daily 1% (w/v)N—Ac-L-Tyr-L-Tyr-OH in WEP442 as prepared in Example 5, to her leftforearm, and a vehicle control WEP442 to her right forearm for 4 weeks.After 2 weeks, there was no change in skin thickness of her rightforearm, her left forearm had increased 16% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. At the end of4 weeks, her vehicle treated right forearm was still loose, relativelythin and wrinkled when lifted. In contrast, her left forearm was morefirm, smooth, plump, and less wrinkled when lifted. While there was nochange in skin thickness of her right forearm, her left forearm hadincreased 17% in skin thickness as measured by the micrometer calipers.This result indicates that N—Ac-L-Tyr-L-Tyr-OH has the potential fortopical treatment of disturbed keratinization and aging related changesof skin, nail and hair.

Example 35

A female subject, age 41, topically applied twice daily 3% (w/v)N—Ac-L-Tyr-L-Tyr-NHNHAc in WEP442 as prepared in Example 5, to her leftforearm, and a vehicle control WEP442 to her right forearm for 6 weeks.After 2 weeks, there was no change in skin thickness of her rightforearm, and her left forearm had increased 7% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. At the end of6 weeks, her vehicle treated right forearm was still loose, relativelythin and wrinkled when lifted. In contrast, her left forearm was morefirm, smooth, plump, and less wrinkled when lifted. While there was nochange in skin thickness of her right forearm, her left forearm hadincreased 23% in skin thickness as measured by the micrometer calipers.This result indicates that N—Ac-L-Tyr-L-Tyr-OH has the potential fortopical treatment of disturbed keratinization and aging related changesof skin, nail and hair.

Example 36

A female subject, age 41, topically applied twice dailyN—Ac-L-Tyr-L-Tyr-OH 1% (w/v) in WEP442 as prepared in Example 5, to herleft forearm, and a vehicle control WEP442 to her right forearm for oneweek. At the end of one week, her vehicle treated right forearm wasstill loose, relatively thin and wrinkled when lifted. In contrast, herleft forearm was more firm, smooth, plump, and less wrinkled whenlifted. While there was no change in skin thickness of her rightforearm, her left forearm had increased 16% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. This resultindicates that N—Ac-L-Tyr-L-Tyr-OH has the potential for topicaltreatment of disturbed keratinization and aging related changes of skin,nail and hair.

Example 37

A female subject, age 59, topically applied twice daily 3% (w/v)N—Ac-L-Tyr-L-Tyr-NH2 in WEP442, pH 6.7 as prepared in Example 5, to herleft forearm, and a vehicle control WEP442 to her right forearm for 4weeks. At the end of 4 weeks, her vehicle treated right forearm wasstill loose, relatively thin and wrinkled when lifted. In contrast, herleft forearm was more firm, smooth, plump, and less wrinkled whenlifted. While there was no change in skin thickness of her rightforearm, her left forearm had increased 21% in skin thickness asmeasured by the micrometer calipers as shown in Example 1. This resultindicates that N—Ac-L-Tyr-L-Tyr-NH2 has the potential for topicaltreatment of disturbed keratinization and aging related changes of skin,nail and hair.

Example 38

A male subject, age 80, had multiple varicose veins in his right lowerleg for many years resistant to any kind of topical treatments includinghigh potency alpha-hydroxyacids. The lesions of varicose veins coveredan area of skin measuring 5 cm×10 cm and appeared more prominent withadvancing age. The subject topically applied twice daily 0.5% (w/v)N—Ac-L-Tyr-L-Tyr-NH₂ in WEP442 solution as prepared in Example 5. At theend of four weeks, the lesions of varicose veins appeared lessprominent. It is believed that continued topical application willeradicate the varicose veins. This result indicates thatN—Ac-L-Tyr-L-Tyr-NH₂ has the potential for topical treatment of varicoseveins.

Example 39

A male subject, age 41, performed pushups routinely with a maximalnumber of about 100 in one hour. The subject topically applied 0.5%(w/v) N—Ac-L-Tyr-L-Tyr-NH2 in WEP442, pH 6.9 solution as prepared inExample 5 to ease the tight muscles on the upper chest about 30 minutesbefore the exercise. The subject was surprised to find that after thetopical application he could now perform pushups with a maximal numberof about 200 in one hour without feeling tired. The subject repeated theperformance next day with the same enhanced result. To make sure thatthe enhanced effect on the muscles was not due to the vehicle solution,in the following day the subject topically applied WEP442 solution tothe same upper chest about 30 minutes before the exercise. This time thesubject could not perform the same number of pushups as before.

The above results show that the bioactive dipeptide derivative of thepresent invention has the potential of enhancing muscle strength andoptimizing exercise performance.

Example 40

A female subject, age 45, having chronic inflammation or eczema withscales, erythema and itch covering large areas of the body for manyyears, failed to respond by conventional treatments including topicalcorticosteroids. The subject topically applied twice daily on one set ofinvolved lesions, 0.6% (w/w) N—Ac-L-Tyr-L-Tyr-NHNH2 cream (Example 4)for two weeks. The itch stopped a few minutes after the topicalapplication. At the end of two weeks, the scales and erythema diminishedsubstantially and the treated skin had 75% improvement as judged byclinical evaluation. The subject also topically applied twice daily onanother set of involved lesions, 0.7% (w/w) N—Ac-L-Tyr-L-Tyr-NHNHAccream (Example 4) for two weeks. The itch stopped immediately after thetopical application. At the end of two weeks, the scales and erythemadisappeared completely, and the treated skin was smooth and appearednormal. The treated lesions had 100% improvement as judged by clinicalevaluation. The results show that the dipeptide derivatives of thepresent invention have the potential for topical treatment of symptomsor syndromes associated with nerve disorders, disturbed keratinization,inflammation and deranged immune system.

Example 41

A male subject, age 90, had severe arthritis of both knees with edemaand pain for many years. The conventional therapy had provided onlytemporary relief of knee pain and edema. The subject tookN—Ac-L-Pro-Gly-NH₂ (1 mg) sublingually and kept the dipeptide under thetongue for 15 minutes before swallowing. After a few hours, the edemaand pain diminished substantially. The above result shows that thedipeptide derivative of the present invention has the potential oftreating inflammation, arthritis, pain, other immune and nerve disordersvia systemic administration.

Example 42

A male subject, age 80, had fungal infections of two toe nails for manyyears. Topical treatments with clotrimazole and salicylic acid with orwithout glycolic acid had not been successful, as shown by thepersistent appearance of white and sandy nail plates. The subjecttopically applied twice daily to one infected nail plate, 0.5% (w/v)N—Ac-L-Tyr-L-Tyr-NH₂ in WEP442, pH 6.7 (Example 5), and to anotherinfected nail plate, 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NHNHAc in WEP442, pH5.5 (Example 5) for several months. After several weeks of topicalapplications, the white and sandy appearance of both nail plates startedto change gradually. Continued topical applications of the formulationsshould eradicate the nail infections, and the toe nails should start togrow normally. The above result shows that the dipeptide derivatives ofthe present invention have the potential of treating nail infections andother infections caused by microorganisms.

Example 43

A female subject, age 73, had multiple age spots including lentiginesand keratosis on her face. Topical treatments with conventionalmedications including hydroquinone with or without glycolic acid hadbeen unsuccessful. The subject topically applied twice daily to the leftside of her face 1% (w/v) N—Ac-L-Cys-L-Tyr-NH2 in WEP442, pH 6.4(Example 5), and to the right side of her face 0.4% (w/v)N—Ac-L-Cys-L-Cys-NH2 in WEP442 (Example 5) for several months. After 4weeks of topical applications, the age spot lesions on both sides of herface started to improve noticeably as shown by the lighter color inappearance. Continued topical applications of the formulations shouldimprove or eradicate lentigines and keratosis of the face. The aboveresult shows that the dipeptide derivatives of the present inventionhave the potential of treating aging related skin changes including agespots and also for skin lightening.

Example 44

A male subject, age 41, routinely took a shower every morning, thentopically applied a conventional antiperspirant product on the underarmskin to prevent perspiration. To test the efficacy of theN-acyldipeptide derivative of the present invention, the subject afterthe shower, topically applied 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH₂ in WEP442,pH 6.7 (Example 5) on the left underarm and a control vehicle WEP442 onthe right underarm. After two hours, there was profuse sweating in theright underarm as evidenced by the wet shirt in the armpit area. Incontrast, there was no sweating in the left underarm as shown by the dryshirt in the armpit area. The left underarm was dry for the next 6-8hours. The above result shows that N—Ac-Tyr-Tyr-NH₂ has the potentialfor topical application to reduce or prevent perspiration or sweating ofunderarm, crotch, palm, or other parts of the body.

Example 45

Typical examples of improving eye vision are described as follows.

A male subject, age 41, wore eyeglasses in both eyes fornear-sightedness (myopia), and had a routine eye checkup every year.Each time his vision in both eyes got worse, and he required changes incontact lenses in past years. This time, 6 weeks before his checkup, thesubject topically applied twice daily 0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH₂ inWEP442, pH 6.7 (Example 5) on the skin around and outside of eye areasand periorbital regions for 6 weeks. During the checkup, the subject andthe optometrist were surprised to find that vision in both eyes did notworsen this time, and there was no need to change the prescription forhis eyeglasses. The above result shows that N—Ac-Tyr-Tyr-NH₂ has thepotential for topical application to improve eye vision.

A male subject, age 43, wore contact lenses in both eyes fornear-sightedness (myopia), and had a routine eye checkup every year.Each time his vision in both eyes worsened, and he required changes inthe prescription for his contact lenses in the past years. This time,one month before his checkup, the subject topically applied once daily0.5% (w/v) N—Ac-L-Tyr-L-Tyr-NH2 in WEP442, pH 6.7 (Example 5) on theskin around and outside of eye areas and periorbital regions for 4weeks. During the checkup, the subject and the optometrist weresurprised to find that vision in both eyes did not worsen this time, andthere was no need to change the prescription for his contact lenses. Theabove result shows that N—Ac-Tyr-Tyr-NH2 has the potential for topicalapplication to improve eye vision.

Example 46

A typical in vitro screen for anti-tumor or anti-cancer effects wascarried out as follows.

An aliquot of 2,000 MB231 breast cancer cells (breast carcinoma cells)in 100 μl DMEM complete media (Sigma Chemical Co.) was plated in a 96well plate containing at a concentration of 20 μg/ml a test substance orcontrol water. To measure proliferation of the cancer cells, an aliquotof 20 μl of MTS reagent (Promega Co.) was added to each well and thecells were incubated at 370C for a total of three days. The cellsrapidly metabolized MTS reagent and the metabolized MTS reagent wasmeasured at 490 nm at the end of days 1, 2 and 3. The reading wasproportional to the number of cancer cells. The decrease in absorbanceat each time point indicated fewer cancer cells. At the end of day 3,the decrease in absorbance with respect to the control indicated theinhibition by the test substance. The test showed thatN—Ac-L-Val-L-Ala-NH2 had inhibited the growth of breast cancer cells byapproximately 12% at the end of 3 days of incubation. The above resultshows that N—Ac-L-Val-L-Ala-NH₂ has the potential for treatment ofbreast cancers.

Supplementary Test Results and Summary

In addition to the above Examples, additional test results and summaryare described in the following sections.

In one embodiment, a composition for topical application to test itstherapeutic effect was formulated by dissolving a bioactive dipeptidederivative of the present invention in a solution prepared from 40 partswater, 40 parts ethanol and 20 parts propylene glycol by volume(WEP442).

In another embodiment, a composition was formulated by dissolving abioactive dipeptide derivative of the present invention in a non-aqueoussolution prepared from 80 parts ethanol and 20 parts propylene glycol byvolume (EP82).

In yet another embodiment, a composition was formulated first bydissolving a bioactive dipeptide derivative of the present invention ina solution prepared from 80 parts water and 20 parts propylene glycol byvolume (WP82), and the solution thus prepared was mixed with anoil-in-water emulsion or cream.

The concentration of the bioactive ingredient in test solution or creamgenerally can range from about 0.001% to about 99.9%, with preferredconcentration of about 0.01% to 30%, with more preferred concentrationof about 0.1% to 10% by weight or by volume (solution composition) ofthe total composition.

As an illustration, 0.4 g of N—Ac-L-Val-L-Ala-NH2 as a white powder wasdissolved in a solution prepared from 40 parts water, 40 parts ethanoland 20 parts propylene glycol by volume (WEP442), and the total volumeof the solution was made up to 100 ml. The solution composition thusformulated had pH 5.8 and contained 0.4% N—Ac-L-Val-L-Ala-NH2 in WEP442.Under the same conditions, test solution compositions containing variousN-acyldipeptide derivatives of the present invention were prepared inWEP442 with concentrations ranging from 0.1% to 10%.

As an another illustration, 1.0 g of N—Ac-L-Tyr-L-Tyr-NH₂ as a whitepowder was dissolved in a solution prepared from 80 parts ethanol and 20parts propylene glycol or 80 parts 95% alcohol and 20 parts propyleneglycol by volume (WEP4:76:20), and the total volume of the solution wasmade up to 100 ml. The solution composition thus formulated contained 1%N—Ac-L-Tyr-L-Tyr-NH₂ in EP82 or WEP4:76:20 respectively. Under the sameconditions, test solution compositions containing variousN-acyldipeptide derivatives of the present invention were prepared inEP82 or WEP with concentrations ranging from 0.1% to 10%.

As a further illustration, 0.3 g of N—Ac-L-Val-L-Ala-NH2 as white powderwas dissolved in 30 ml warm solution prepared from 80 parts water and 20parts propylene glycol by volume. The solution thus prepared was mixedwith 69.7 g oil-in-water emulsion or cream. The composition thusformulated contained 0.3% N—Ac-L-Val-L-Ala-NH2 in an emulsion or creamcomposition. Under the same conditions, oil-in-water or creamcompositions containing various N-acyldipeptide derivatives of thepresent invention were formulated with concentrations ranging from 0.1%to 10% by weight of the total composition.

A test composition containing a bioactive dipeptide derivative of thepresent invention for systemic administration was prepared according toExample 22.

For injection administration, a composition was prepared with or withouta thickening agent, such as methyl cellulose. As an illustration,N—Ac-L-Tyr-L-Tyr-NH2, 100 mg, was dissolved in 5 ml water, and thesolution was sterizilized in an injection vial. The compositions thusprepared contained 2% (w/w) or 20 mg/ml N—Ac-L-Tyr-L-Tyr-NH2 suitablefor intra-articular, intra-lesion, or subcutaneous injection, or othersystemic administration.

Under the same conditions, the following compositions for systemicadministration were prepared: 2% or 20 mg/ml N—Ac-L-Tyr-L-Tyr-NHNH₂ inwater with or without 1% methyl cellulose; 2% or 20 mg/mlN—Ac-L-Tyr-L-Tyr-NHNHAc in water with or without 1% methyl cellulose;0.2% or 2 mg/ml N—Ac-L-Val-L-Ala-NH₂ in water with or without 1% methylcellulose; and 0.2% or 2 mg/ml N—Ac-L-Tyr-L-Tyr-OH in water with orwithout 1% methyl cellulose.

Volunteer Subjects:

In these studies, the participating subjects were as follows:

Subject 1. Male, age 78, had multiple red and itchy inflammation,dermatitis, or eczema lesions, which were resistant to conventionaltreatments including corticosteroids.Subject 2. Female, age 31, had small red and itchy lesions which wereresistant to topical corticosteroid treatment.Subject 3. Female, age 43, had multiple red and itchy inflammation,dermatitis, or eczema lesions over the body for many years, which wereresistant to conventional treatments including corticosteroids.Subject 4. Female, age 50, had red and itchy inflammation, dermatitis,or eczema lesions for many years, which were resistant to topicalcorticosteroid treatment.Subject 5. Female, age 41, had early stage of aging related skin changeson both forearms as indicated by age spots and wrinkled skin caused bysolar damage.Subject 6. Female, age 52, had age spots, keratoses and wrinkles on bothforearms caused by intrinsic and extrinsic aging.Subject 7. Female, age 51, had age spots, and wrinkles on both forearmscaused by intrinsic and extrinsic aging.Subject 8. Male, age 90, had osteoarthritis of both knees withinflammation and pain for more than 4 years, and had only mildtransitory relief from conventional treatments.Subject 9. Female, age 41, had sensitive skin with inflammatory lesionson the body.Subject 10. Male, age 42, had mosquito bites and inflammatory lesions onthe body.Subject 11. Male, age 41, had stretch marks all over the body due tomassive weight loss.Subject 12. Male, age 41, had dermatitis and inflammatory lesions on theleft palm.Subject 13. Female, age 52, had chigger bites with multiple lesions onher legs.Subject 14. Female, age 73, had multiple age spots including lentiginesand keratosis on her face.

Other subjects with various skin and medical conditions and disordersalso participated in the present tests and studies.

Test Methods.

In one embodiment, the test compositions containing N-acyldipeptidederivatives of the present invention were tested in an in vitro screenfor their biological efficacy in cell cultures as described in Examples21 and 46.

In another embodiment, the volunteer subject topically applied the testcompositions containing N-acyldipeptide derivatives of the presentinvention on involved skin or lesions once or twice daily for severalweeks or until the involved lesions completely cleared and clinicallychanged to normal skin. As a control study, the subject also topicallyapplied a vehicle control composition on the involved skin or lesionstwice daily for the same period.

In yet another embodiment, the volunteer subject topically applied onceor twice daily the test compositions containing N-acyldipeptidederivatives of the present invention on the skin site above arthriticjoints or painful muscles to provide therapeutic effects for thesystemic disorders via topical administration.

In yet another embodiment, the volunteer subject injectedintra-articularly into a knee joint a test composition containing abioactive dipeptide derivative of the present invention to improve andreduce arthritic inflammation and pain of the joint.

Some test results are summarized as follows.

DK: Disturbed keratinizationAG: Aging related changes of skin, nail and hairDI: Deranged immune disorders and inflammationND: Nerve disorders

Ala Peptide Derivative DK AG DI ND N-Ac-L-Ile-L-Ala-NH₂ 2+ 2+ 4+ 4+N-Ac-L-Ile-L-Ala-OH 2+ 2+ 2+ 2+ N-Ac-L-Leu-L-Ala-NH₂ 3+ 3+ 4+ 4+N-Ac-L-Leu-L-Ala-OH 2+ 2+ 3+ 3+ N-Ac-L-Val-L-Ala-NH₂ 4+ 4+ 4+ 4+ alsofor joints, muscles, breast cancer N-Ac-L-Val-L-Ala-OH 2+ 2+ 3+ 4+N-Pr-L-Val-L-Ala-OH 2+ 2+ 3+ 4+ N-Ac-L-Cys-L-Cys-NH₂ 2+ 3+ 2+ 2+ alsofor age spots N-Ac-L-Cys-L-Cys-OH 2+ 3+ 2+ 2+ also for age spotsN-Ac-β-Ala-Gly-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Asn-Gly-NH₂ 2+ 2+ 2+ 2+N-Ac-Abz-Gly-NH₂ 2+ 2+ 3+ 3+ N-Ac-L-Cys-Gly-NH₂ 2+ 2+ 2+ 2+N-Ac-Gaba-Gly-NH₂ 2+ 2+ 2+ 2+ N-Ac-Gly-Gly-NH₂ 2+ 2+ 2+ 2+N-Ac-L-Gln-Gly-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-His-Gly-NH₂ 2+ 2+ 2+ 2+N-Ac-L-Ile-Gly-NH₂ 3+ 3+ 4+ 4+ N-Ac-L-Ile-Gly-OH 2+ 2+ 3+ 4+N-Ac-L-Leu-Gly-NH₂ 3+ 3+ 4+ 4+ N-Ac-L-Leu-Gly-OH 2+ 2+ 3+ 3+N-Pr-L-Leu-Gly-OH 2+ 2+ 3+ 3+ N-Ac-L-Pro-Gly-NH₂ 2+ 2+ 3+ 4+N-Ac-L-Ser-Gly-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Tyr-Gly-NH₂ 2+ 2+ 2+ 2+N-Z-L-Tyr-Gly-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Val-Gly-NH₂ 2+ 2+ 4+ 4+N-Ac-L-Val-Gly-OH 2+ 2+ 3+ 4+ N-Ac-βAla-L-His-NH₂ 2+ 2+ 2+ 2+N,N′-diAc-βAla-L-His-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Ala-L-Ile-NH₂ 2+ 2+ 3+ 3+N-Ac-L-Ile-L-Ile-NH₂ 3+ 2+ 3+ 4+ N-Ac-L-Leu-L-Leu-NH₂ 2+ 2+ 2+ 2+N-Ac-Gly-L-Pro-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Cys-L-Tyr-NH₂ 2+ 2+ 2+ 3+N-Ac-L-Ile-L-Tyr-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Leu-L-Tyr-NH₂ 2+ 2+ 2+ 2+N-Ac-L-Tyr-L-Tyr-NH₂ 4+ 4+ 4+ 4+ also for joints, muscles, infectionsN-Ac-L-Val-L-Tyr-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Tyr-L-Tyr-OH 3+ 4+ 3+ 4+N-Ac-L-Tyr-L-Tyr-OEt 2+ 2+ 2+ 2+ N-Ac-L-Tyr-L-Tyr-NHNH₂ 3+ 3+ 2+ 2+N-Ac-L-Tyr-L-Tyr- 4+ 3+ 4+ 4+ also for NHNHAc infectionsN-Ac-L-Tyr-L-Hpg-NH₂ 2+ 2+ 2+ 2+ N-Ac-L-Phe-L-Phe-NH₂ 2+ 2+ 2+ 2+N-Ac-L-Trp-L-Trp-NH₂ 2+ 2+ 2+ 2+ 1+: 25% Efficacy 2+: 50% Efficacy 3+:75% Efficacy 4+: 95-100% Efficacy

The bioactive dipeptide derivatives of the present invention can betopically administered to provide topical effects or to exerttherapeutic effects for systemic diseases. As shown in Examples 27, 31,32, 39, 44 and 45, the compositions containing N-acyldipeptidederivatives have the potential to improve arthritis and pain of joints,enhance muscle strength, improve eye vision and to reduce or preventsweating or perspiration of underarm, crotch, palm, or other parts ofthe body, via topical application. The bioactive dipeptide derivativesof the present invention can also be given by systemic administration toimprove systemic diseases. As shown in the Example 23, the compositioncontaining the bioactive dipeptide derivative has the potential toimprove arthritis of knee joints via intra-articular injection.

The increased skin thickness or plump as shown in the Examples was notdue to increased water retention or edema of the skin because thethickness maintained for many months after discontinuation of thetreatment. As shown earlier, the skin biopsies from the plump skin showincreased biosynthesis of GAGs, collagen fibers and elastic fibers, andless clumping of melanin as evidenced by histochemical analysis.Therefore, when a substance is found to plump or increase the skinthickness, the substance is reasonably expected or predicted to improveaging related skin changes including fine lines, wrinkles, photoaging,age spots, blotches, hyperpigmented skin, mottled skin, and foryounger-looking skin and skin lightening.

Therefore, while not wishing to be bound by theory, it is believed thata composition of the present invention has the potential for topicaltreatment of aging related skin changes including wrinkles andolder-looking skin, because a bioactive dipeptide derivative canincrease the skin thickness or plump the skin.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

We claim:
 1. A composition comprising gluconolactone and atherapeutically effective amount of a dipeptide derivative of formula(I):R₁-AAB-AAC-R₂  Formula (I) or an isomer or salt thereof, and optionallya pharmaceutically or cosmetically acceptable carrier, wherein R₁ is anacyl radical having up to 9 carbon atoms; AAB is Tyr and AAC is Tyr, orAAB is Val and AAC is Ala; R₂ is OR₃, NHR₄, or NHNHR₅; R₃ is H, analkyl, aralkyl, or aryl radical having up to 19 carbon atoms; and R₄ orR₅ is independently H, OH, an alkyl, aralkyl, or aryl radical having upto 19 carbon atoms.
 2. The composition of claim 1, wherein AAB is Tyrand AAC is Tyr.
 3. The composition of claim 1, wherein AAB is Val andAAC is Ala.
 4. The composition of claim 1, wherein the dipeptidederivative is selected from the group consisting of N—Ac-Tyr-Tyr-NH₂,N—Ac-Tyr-Tyr-OH, N—Ac-Tyr-Tyr-OEt, N—Ac-Tyr-Tyr-NHNH₂,N—Ac-Tyr-Tyr-NHNHAc, N—Ac-Tyr-Tyr-NHOH, N—Pr-Tyr-Tyr-NH₂,N—Pr-Tyr-Tyr-OH, N—Pr-Tyr-Tyr-OEt, N—Pr-Tyr-Tyr-NHNH₂, andN—Pr-Tyr-Tyr-NHNHPr.
 5. The composition of claim 1, wherein thedipeptide derivative is selected from the group consisting ofN—Ac-Val-Ala-NH₂, N—Ac-Val-Ala-OH, N—Ac-Val-Ala-NHOH, N—Pr-Val-Ala-NH₂,and N—Pr-Val-Ala-OH.
 6. The composition of claim 1, wherein thedipeptide derivative is selected from the group consisting ofN—Ac-Tyr-Tyr-NH₂ and N—Ac-Val-Ala-NH₂.
 7. The composition of claim 1,wherein the dipeptide derivative is N—Ac-Val-Ala-NH₂.
 8. A method oftreating aging related skin changes or changes associated with intrinsicor extrinsic aging, or of increasing skin thickness in a subject in needthereof, the method comprising topically administering to skin of thesubject the composition of claim
 1. 9. The method of claim 8, whereinthe dipeptide derivative is selected from the group consisting ofN—Ac-Tyr-Tyr-NH₂, N—Ac-Tyr-Tyr-OH, N—Ac-Tyr-Tyr-OEt, N—Ac-Tyr-Tyr-NHNH₂,N—Ac-Tyr-Tyr-NHNHAc, N—Ac-Tyr-Tyr-NHOH, N—Pr-Tyr-Tyr-NH₂,N—Pr-Tyr-Tyr-OH, N—Pr-Tyr-Tyr-OEt, N—Pr-Tyr-Tyr-NHNH₂, andN—Pr-Tyr-Tyr-NHNHPr.
 10. The method of claim 8, wherein the dipeptidederivative is selected from the group consisting of N—Ac-Val-Ala-NH₂,N—Ac—Val-Ala-OH, N—Ac—Val-Ala-NHOH, N—Pr—Val-Ala-NH₂, andN—Pr-Val-Ala-OH.
 11. The method of claim 8, wherein the dipeptidederivative is selected from the group consisting of N—Ac-Tyr-Tyr-NH₂ andN—Ac-Val-Ala-NH₂.
 12. The method of claim 8, wherein the treatment isfor aging related skin changes selected from the group consisting offine lines; wrinkles; age spots; blotches; lentigines; mottled skin;laxity; photoaging; photodamage; hyperpigmentation; stretch marks;thinning of skin; cellulite; elastosis; loss or reduction of skinresiliency, elasticity, and recoilability; and abnormal or diminishedsynthesis of collagen, glycosaminoglycans, proteoglycans, or elastin.13. The method of claim 8, wherein the treatment is for aging relatedskin changes selected from the group consisting of fine lines; wrinkles;laxity; hyperpigmentation; age spots; lentigines; mottled skin;cellulite; and photoaging.
 14. A method of treating fine lines,wrinkles, laxity, hyperpigmentation, or photoaging in a subject in needthereof, the method comprising topically administering to skin of thesubject the composition of claim
 1. 15. The method of claim 14, whereinthe dipeptide derivative is selected from the group consisting ofN—Ac-Tyr-Tyr-NH₂, N—Ac-Tyr-Tyr-OH, N—Ac-Tyr-Tyr-OEt, N—Ac-Tyr-Tyr-NHNH₂,N—Ac-Tyr-Tyr-NHNHAc, N—Ac-Tyr-Tyr-NHOH, N—Pr-Tyr-Tyr-NH₂,N—Pr-Tyr-Tyr-OH, N—Pr-Tyr-Tyr-OEt, N—Pr-Tyr-Tyr-NHNH₂,N—Pr-Tyr-Tyr-NHNHPr, N—Ac—Val-Ala-OH, N—Ac—Val-Ala-NHOH,N—Pr—Val-Ala-NH₂, and N—Pr-Val-Ala-OH, or an isomer or salt thereof, andoptionally a pharmaceutically or cosmetically acceptable carrier. 16.The method of claim 14, wherein the dipeptide derivative is selectedfrom the group consisting of N—Ac-Tyr-Tyr-NH₂ and N—Ac-Val-Ala-NH₂. 17.A method of treating aging related skin changes or changes associatedwith intrinsic or extrinsic aging, or of increasing skin thickness in asubject in need thereof, the method comprising topically administeringto skin of the subject a composition comprising gluconolactone and atherapeutically effective amount of N—Ac-Val-Ala-NH₂, or an isomer orsalt thereof, and optionally a pharmaceutically or cosmeticallyacceptable carrier.
 18. The method of claim 17, wherein the treatment isfor aging related skin changes selected from the group consisting offine lines; wrinkles; age spots; blotches; lentigines; mottled skin;laxity; photoaging; photodamage; hyperpigmentation; stretch marks;thinning of skin; cellulite; elastosis; loss or reduction of skinresiliency, elasticity, and recoilability; and abnormal or diminishedsynthesis of collagen, glycosaminoglycans, proteoglycans, or elastin.19. The method of claim 17, wherein the method is for treating finelines, wrinkles, laxity, hyperpigmentation, or photoaging.